Richard M. Mortensen

PPARγ Is Required for the Differentiation of Adipose Tissue In Vivo and In Vitro

Abstract The process of adipogenesis is known to involve the interplay of several transcription factors. Activation of one of these factors, the nuclear hormone receptor PPARγ, is known to promote fat cell differentiation in vitro. Whether PPARγ is required for this process in vivo has remained an open question because a viable loss-of-function model for PPARγ has been lacking. We demonstrate here that mice chimeric for wild-type and PPARγ null cells show little or no contribution of null cells to adipose tissue, whereas most other organs examined do not require PPARγ for proper development. In vitro, the differentiation of ES cells into fat is shown to be dependent on PPARγ gene dosage. These data provide direct evidence that PPARγ is essential for the formation of fat.

The role of PPAR-γ in macrophage differentiation and cholesterol uptake

Peroxisome proliferator-activated receptor-γ (PPAR-γ), the transcription factor target of the anti-diabetic thiazolidinedione (TZD) drugs, is reported to mediate macrophage differentiation and inflammatory responses. Using PPAR-γ–deficient stem cells, we demonstrate that PPAR-γ is neither essential for myeloid development, nor for such mature macrophage functions as phagocytosis and inflammatory cytokine production. PPAR-γ is required for basal expression of CD36, but not for expression of the other major scavenger receptor responsible for uptake of modified lipoproteins, SR-A. In wild-type macrophages, TZD treatment divergently regulated CD36 and class A macrophage-scavenger receptor expression and failed to induce significant cellular cholesterol accumulation, indicating that TZDs may not exacerbate macrophage foam-cell formation.

Production of homozygous mutant ES cells with a single targeting construct.

We have developed a simple method for producing embryonic stem (ES) cell lines whereby both alleles have been inactivated by homologous recombination and which requires a single targeting construct. Four different ES cell lines were created that were heterozygous for genes encoding two guanine nucleotide-binding protein subunits, alpha i2 and alpha i3, T-cell receptor alpha, and beta-cardiac myosin heavy chain. When these heterozygous cells were grown in high concentrations of G418, many of the surviving cells were homozygous for the targeted allele and contained two copies of the G418 resistance gene. This scheme provides an easy method for obtaining homozygous mutationally altered cells, i.e., double knockouts, and should be generally applicable to other genes and to cell lines other than ES cells. This method should also enable the production of cell lines in which more than one gene have had both alleles disrupted. These mutant cells should provide useful tools for defining the role of particular genes in cell culture.

Cardiac-Specific Overexpression of GLUT1 Prevents the Development of Heart Failure Attributable to Pressure Overload in Mice

Background—Increased rates of glucose uptake and glycolysis have been repeatedly observed in cardiac hypertrophy and failure. Although these changes have been considered part of the fetal gene reactivation program, the functional significance of increased glucose utilization in hypertrophied and failing myocardium is poorly understood. Methods and Results—We generated transgenic (TG) mice with cardiac-specific overexpression of insulin-independent glucose transporter GLUT1 to recapitulate the increases in basal glucose uptake rate observed in hypertrophied hearts. Isolated perfused TG hearts showed a greater rate of basal glucose uptake and glycolysis than hearts isolated from wild-type littermates, which persisted after pressure overload by ascending aortic constriction (AAC). The in vivo cardiac function in TG mice, assessed by echocardiography, was unaltered. When subjected to AAC, wild-type mice exhibited a progressive decline in left ventricular (LV) fractional shortening accompanied by ventricular dilation and decreased phosphocreatine to ATP ratio and reached a mortality rate of 40% at 8 weeks. In contrast, TG-AAC mice maintained LV function and phosphocreatine to ATP ratio and had <10% mortality. Conclusions—We found that increasing insulin-independent glucose uptake and glycolysis in adult hearts does not compromise cardiac function. Furthermore, we demonstrate that increasing glucose utilization in hypertrophied hearts protects against contractile dysfunction and LV dilation after chronic pressure overload.

Myeloid mineralocorticoid receptor controls macrophage polarization and cardiovascular hypertrophy and remodeling in mice.

Inappropriate excess of the steroid hormone aldosterone, which is a mineralocorticoid receptor (MR) agonist, is associated with increased inflammation and risk of cardiovascular disease. MR antagonists are cardioprotective and antiinflammatory in vivo, and evidence suggests that they mediate these effects in part by aldosterone-independent mechanisms. Here we have shown that MR on myeloid cells is necessary for efficient classical macrophage activation by proinflammatory cytokines. Macrophages from mice lacking MR in myeloid cells (referred to herein as MyMRKO mice) exhibited a transcription profile of alternative activation. In vitro, MR deficiency synergized with inducers of alternatively activated macrophages (for example, IL-4 and agonists of PPARgamma and the glucocorticoid receptor) to enhance alternative activation. In vivo, MR deficiency in macrophages mimicked the effects of MR antagonists and protected against cardiac hypertrophy, fibrosis, and vascular damage caused by L-NAME/Ang II. Increased blood pressure and heart rates and decreased circadian variation were observed during treatment of MyMRKO mice with L-NAME/Ang II. We conclude that myeloid MR is an important control point in macrophage polarization and that the function of MR on myeloid cells likely represents a conserved ancestral MR function that is integrated in a transcriptional network with PPARgamma and glucocorticoid receptor. Furthermore, myeloid MR is critical for blood pressure control and for hypertrophic and fibrotic responses in the mouse heart and aorta.

Peroxisome Proliferator-Activated Receptor-γ–Mediated Effects in the Vasculature

Peroxisome proliferator-activated receptor (PPAR)-γ is a nuclear receptor and transcription factor in the steroid superfamily. PPAR-γ agonists, the thiazolidinediones, are clinically used to treat type 2 diabetes. In addition to its function in adipogenesis and increasing insulin sensitivity, PPAR-γ also plays critical roles in the vasculature. In vascular endothelial cells, PPAR-γ activation inhibits endothelial inflammation by suppressing inflammatory gene expression and therefore improves endothelial dysfunction. In vascular smooth muscle cells, PPAR-γ activation inhibits proliferation and migration and promotes apoptosis. In macrophages, PPAR-γ activation suppresses inflammation by regulating gene expression and increases cholesterol uptake and efflux. A recurring theme in many cell types is the modulation of the innate immunity system particularly through altering the activity of the nuclear factor &kgr;B. This system is likely to be even more prominent in modulating disease in vascular cells. The effects of PPAR-γ in the vascular cells translate into the beneficial function of this transcription factor in vascular disorders, including hypertension and atherosclerosis. Both human genetic studies and animal studies using transgenic mice have demonstrated the importance of PPAR-γ in these disorders. However, recent clinical studies have raised significant concerns about the cardiovascular side effects of thiazolidinediones, particularly rosiglitazone. Weighing the potential benefit and harm of PPAR-γ activation and exploring the functional mechanisms may provide a balanced view on the clinical use of these compounds and new approaches to the future therapeutics of vascular disorders associated with diabetes.

Cardiomyocyte-Specific Knockout and Agonist of Peroxisome Proliferator–Activated Receptor-γ Both Induce Cardiac Hypertrophy in Mice

Peroxisome proliferator–activated receptor (PPAR)-γ is required for adipogenesis but is also found in the cardiovascular system, where it has been proposed to oppose inflammatory pathways and act as a growth suppressor. PPAR-γ agonists, thiazolidinediones (TZDs), inhibit cardiomyocyte growth in vitro and in pressure overload models. Paradoxically, TZDs also induce cardiac hypertrophy in animal models. To directly determine the role of cardiomyocyte PPAR-γ, we have developed a cardiomyocyte-specific PPAR-γ–knockout (CM-PGKO) mouse model. CM-PGKO mice developed cardiac hypertrophy with preserved systolic cardiac function. Treatment with a TZD, rosiglitazone, induced cardiac hypertrophy in both littermate control mice and CM-PGKO mice and activated distinctly different hypertrophic pathways from CM-PGKO. CM-PGKO mice were found to have increased expression of cardiac embryonic genes (atrial natriuretic peptide and β-myosin heavy chain) and elevated nuclear factor &kgr;B activity in the heart, effects not found by rosiglitazone treatment. Rosiglitazone increased cardiac phosphorylation of p38 mitogen-activated protein kinase independent of PPAR-γ, whereas rosiglitazone induced phosphorylation of extracellular signal–related kinase 1/2 in the heart dependent of PPAR-γ. Phosphorylation of c-Jun N-terminal kinases was not affected by rosiglitazone or CM-PGKO. Surprisingly, despite hypertrophy, Akt phosphorylation was suppressed in CM-PGKO mouse heart. These data show that cardiomyocyte PPAR-γ suppresses cardiac growth and embryonic gene expression and inhibits nuclear factor &kgr;B activity in vivo. Further, rosiglitazone causes cardiac hypertrophy at least partially independent of PPAR-γ in cardiomyocytes and through different mechanisms from CM-PGKO.

Activation of Rap1B by Gi Family Members in Platelets

It has become increasingly appreciated that receptors coupled to Gαi family members can stimulate platelet aggregation, but the mechanism for this has remained unclear. One possible mediator is the small GTPase, Rap1, which has been shown to contribute to integrin activation in several cell lines and to be activated by a calcium-dependent mechanism in platelets. Here, we demonstrate that Rap1 is also activated by Gαi family members in platelets. First, we show that platelets from mice lacking the Gαi family member Gαz (which couples to the α2A adrenergic receptor) are deficient in epinephrine-stimulated Rap1 activation. We also show that platelets from mice lacking Gαi2, which couples to the ADP receptor, P2Y12, exhibit reduced Rap1 activation in response to ADP. In contrast, platelets from mice that lack Gαq show no decrease in the ability to activate Rap1 in response to epinephrine but show a partial reduction in ADP-stimulated Rap1 activation. This result, combined with studies of human platelets treated with ADP receptor-selective inhibitors, indicates that ADP-stimulated Rap1 activation in human platelets is dependent on both the Gαi-coupled P2Y12 receptor and the Gαq-coupled P2Y1 receptor. Gαi-dependent activation of Rap1 in platelets does not appear to be mediated by enhanced intracellular calcium release because no increase in intracellular calcium concentration was detected in response to epinephrine and because the calcium response to ADP was not diminished in platelets from the Gαi2−/− mouse. Finally, using human platelets treated with selective inhibitors of phosphatidylinositol 3-kinase (PI3K) and mouse platelets selectively lacking the Gβγ-activated form of his enzyme (PI3Kγ), we show that Gi-mediated Rap1 activation is PI3K-dependent. In summary, activation of Rap1 can be stimulated by Gαi- and PI3K-dependent mechanisms in platelets and by Gq- and Ca2+-dependent mechanisms, both of which may play a role in promoting platelet activation.

β-Arrestin1 Knockout Mice Appear Normal but Demonstrate Altered Cardiac Responses to β-Adrenergic Stimulation

Abstract β-Arrestin1 knockout mice were studied to define the physiological role of β-arrestin1 in the regulation of G protein–coupled receptors. β-Arrestin1 is thought to be involved in the desensitization of many G protein–associated cell surface receptors, particularly β-adrenergic receptors. Homozygous knockout mice are overtly normal. Resting cardiovascular parameters modulated by β-adrenergic receptors such as heart rate, blood pressure, and left ventricular ejection fraction are not changed. However, homozygous mutants are more sensitive to β-receptor agonist–stimulated increases in ejection fraction, consistent with a role of β-arrestin1 in β-adrenergic receptor desensitization. We conclude that β-arrestin1 is important for in vivo G protein–coupled receptor desensitization and that this aspect of desensitization represents a mechanism for fine-tuning responses. However, β-arrestin1 does not appear to be required for development or for other essential biological functions.

Hypotension, lipodystrophy, and insulin resistance in generalized PPARγ-deficient mice rescued from embryonic lethality

We rescued the embryonic lethality of global PPARgamma knockout by breeding Mox2-Cre (MORE) mice with floxed PPARgamma mice to inactivate PPARgamma in the embryo but not in trophoblasts and created a generalized PPARgamma knockout mouse model, MORE-PPARgamma knockout (MORE-PGKO) mice. PPARgamma inactivation caused severe lipodystrophy and insulin resistance; surprisingly, it also caused hypotension. Paradoxically, PPARgamma agonists had the same effect. We showed that another mouse model of lipodystrophy was hypertensive, ruling out the lipodystrophy as a cause. Further, high salt loading did not correct the hypotension in MORE-PGKO mice. In vitro studies showed that the vasculature from MORE-PGKO mice was more sensitive to endothelial-dependent relaxation caused by muscarinic stimulation, but was not associated with changes in eNOS expression or phosphorylation. In addition, vascular smooth muscle had impaired contraction in response to alpha-adrenergic agents. The renin-angiotensin-aldosterone system was mildly activated, consistent with increased vascular capacitance or decreased volume. These effects are likely mechanisms contributing to the hypotension. Our results demonstrated that PPARgamma is required to maintain normal adiposity and insulin sensitivity in adult mice. Surprisingly, genetic loss of PPARgamma function, like activation by agonists, lowered blood pressure, likely through a mechanism involving increased vascular relaxation.