Karen Reue

Lipodystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin

Mice carrying mutations in the fatty liver dystrophy (fld) gene have features of human lipodystrophy, a genetically heterogeneous group of disorders characterized by loss of body fat, fatty liver, hypertriglyceridemia and insulin resistance. Through positional cloning, we have isolated the gene responsible and characterized two independent mutant alleles, fld and fld2J. The gene (Lpin1) encodes a novel nuclear protein which we have named lipin. Consistent with the observed reduction of adipose tissue mass in fld and fld 2J mice, wild-type Lpin1 mRNA is expressed at high levels in adipose tissue and is induced during differentiation of 3T3-L1 pre-adipocytes. Our results indicate that lipin is required for normal adipose tissue development, and provide a candidate gene for human lipodystrophy. Lipin defines a novel family of nuclear proteins containing at least three members in mammalian species, and homologs in distantly related organisms from human to yeast.

Lamins A and C but Not Lamin B1 Regulate Nuclear Mechanics

Mutations in the nuclear envelope proteins lamins A and C cause a broad variety of human diseases, including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy, and Hutchinson-Gilford progeria syndrome. Cells lacking lamins A and C have reduced nuclear stiffness and increased nuclear fragility, leading to increased cell death under mechanical strain and suggesting a potential mechanism for disease. Here, we investigated the contribution of major lamin subtypes (lamins A, C, and B1) to nuclear mechanics by analyzing nuclear shape, nuclear dynamics over time, nuclear deformations under strain, and cell viability under prolonged mechanical stimulation in cells lacking both lamins A and C, cells lacking only lamin A (i.e. “lamin C-only” cells), cells lacking wild-type lamin B1, and wild-type cells. Lamin A/C-deficient cells exhibited increased numbers of misshapen nuclei and had severely reduced nuclear stiffness and decreased cell viability under strain. Lamin C-only cells had slightly abnormal nuclear shape and mildly reduced nuclear stiffness but no decrease in cell viability under strain. Interestingly, lamin B1-deficient cells exhibited normal nuclear mechanics despite having a significantly increased frequency of nuclear blebs. Our study indicates that lamins A and C are important contributors to the mechanical stiffness of nuclei, whereas lamin B1 contributes to nuclear integrity but not stiffness.

UCP2 regulates energy metabolism and differentiation potential of human pluripotent stem cells

It has been assumed, based largely on morphologic evidence, that human pluripotent stem cells (hPSCs) contain underdeveloped, bioenergetically inactive mitochondria. In contrast, differentiated cells harbour a branched mitochondrial network with oxidative phosphorylation as the main energy source. A role for mitochondria in hPSC bioenergetics and in cell differentiation therefore remains uncertain. Here, we show that hPSCs have functional respiratory complexes that are able to consume O2 at maximal capacity. Despite this, ATP generation in hPSCs is mainly by glycolysis and ATP is consumed by the F1F0 ATP synthase to partially maintain hPSC mitochondrial membrane potential and cell viability. Uncoupling protein 2 (UCP2) plays a regulating role in hPSC energy metabolism by preventing mitochondrial glucose oxidation and facilitating glycolysis via a substrate shunting mechanism. With early differentiation, hPSC proliferation slows, energy metabolism decreases, and UCP2 is repressed, resulting in decreased glycolysis and maintained or increased mitochondrial glucose oxidation. Ectopic UCP2 expression perturbs this metabolic transition and impairs hPSC differentiation. Overall, hPSCs contain active mitochondria and require UCP2 repression for full differentiation potential.

Three Mammalian Lipins Act as Phosphatidate Phosphatases with Distinct Tissue Expression Patterns

We previously identified mutations in the Lpin1 gene, encoding lipin-1, as the underlying cause of lipodystrophy in the fatty liver dystrophy (fld) mutant mouse. Lipin-1 is normally expressed at high levels in adipose tissue and skeletal muscle, and deficiency in the fld mouse causes impaired adipose tissue development, insulin resistance, and altered energy expenditure. We also identified two additional lipin protein family members of unknown function, lipin-2 and lipin-3. Han et al. (Han, G. S., Wu, W. I., and Carman, G. M. (2006) J. Biol. Chem. 281, 9210–9218) recently demonstrated that the single lipin homolog in yeast, Smp2, exhibits phosphatidate phosphatase type-1 (PAP1) activity, which has a key role in glycerolipid synthesis. Here we demonstrate that lipin-1 accounts for all of the PAP1 activity in white and brown adipose tissue and skeletal muscle. However, livers of lipin-1-deficient mice exhibited normal PAP1 activity, indicating that other members of the lipin protein family could have PAP1 activity. Consistent with this possibility, recombinant lipin-2 and lipin-3 possess PAP1 activity. Each of the three lipin family members showed Mg2+-dependent activity that was specific for phosphatidate under the conditions employed. The different lipins showed distinct tissue expression patterns. Our results establish the three mammalian lipin proteins as PAP1 enzymes and explain the biochemical basis for lipodystrophy in the lipin-1-deficient fld mouse.

Biochemistry, physiology, and genetics of GPAT, AGPAT, and lipin enzymes in triglyceride synthesis

Triacylglycerol (TAG) synthesis and storage in tissues such as adipose tissue and liver have important roles in metabolic homeostasis. The molecular identification of genes encoding enzymes that catalyze steps in TAG biosynthesis from glycerol 3-phosphate has revealed an unexpected number of protein isoforms of the glycerol phosphate acyltransferase (GPAT), acylglycerolphosphate acyltransferase (AGPAT), and lipin (phosphatidate phosphatase) families that appear to catalyze similar biochemical reactions. However, on the basis of available data for a few members in which genetic deficiencies in mouse and/or human have been studied, we postulate that each GPAT, AGPAT, and lipin family member likely has a specialized role that may be uncovered through careful biochemical and physiological analyses.

Metabolic syndrome: from epidemiology to systems biology

Metabolic syndrome (MetSyn) is a group of metabolic conditions that occur together and promote the development of cardiovascular disease (CVD) and diabetes. Recent genome-wide association studies have identified several novel susceptibility genes for MetSyn traits, and studies in rodent models have provided important molecular insights. However, as yet, only a small fraction of the genetic component is known. Systems-based approaches that integrate genomic, molecular and physiological data are complementing traditional genetic and biochemical approaches to more fully address the complexity of MetSyn.

Metabolic state of glioma stem cells and nontumorigenic cells

Gliomas contain a small number of treatment-resistant glioma stem cells (GSCs), and it is thought that tumor regrowth originates from GSCs, thus rendering GSCs an attractive target for novel treatment approaches. Cancer cells rely more on glycolysis than on oxidative phosphorylation for glucose metabolism, a phenomenon used in 2-[18F]fluoro-2-deoxy-d-glucose positron emission tomography imaging of solid cancers, and targeting metabolic pathways in cancer cells has become a topic of considerable interest. However, if GSCs are indeed important for tumor control, knowledge of the metabolic state of GSCs is needed. We hypothesized that the metabolism of GSCs differs from that of their progeny. Using a unique imaging system for GSCs, we assessed the oxygen consumption rate, extracellular acidification rate, intracellular ATP levels, glucose uptake, lactate production, PKM1 and PKM2 expression, radiation sensitivity, and cell cycle duration of GSCs and their progeny in a panel of glioma cell lines. We found GSCs and progenitor cells to be less glycolytic than differentiated glioma cells. GSCs consumed less glucose and produced less lactate while maintaining higher ATP levels than their differentiated progeny. Compared with differentiated cells, GSCs were radioresistant, and this correlated with a higher mitochondrial reserve capacity. Glioma cells expressed both isoforms of pyruvate kinase, and inhibition of either glycolysis or oxidative phosphorylation had minimal effect on energy production in GSCs and progenitor cells. We conclude that GSCs rely mainly on oxidative phosphorylation. However, if challenged, they can use additional metabolic pathways. Therefore, targeting glycolysis in glioma may spare GSCs.

Alternatively Spliced Lipin Isoforms Exhibit Distinct Expression Pattern, Subcellular Localization, and Role in Adipogenesis

We recently identified mutations in the Lpin1 (lipin) gene to be responsible for lipodystrophy in the fatty liver dystrophy (fld) mouse strain. Previous studies revealed that lipin plays a critical role in adipogenesis, explaining the adipose-deficient phenotype of the fld mouse. In the current study, we demonstrate that alternative mRNA splicing generates two lipin isoforms, lipin-α and lipin-β, which are differentially expressed during adipocyte differentiation. Lipin-α expression peaks at day 2 of 3T3-L1 cell differentiation, after which its levels gradually decrease. In contrast, lipin-β expression is transiently elevated at 10 h, followed by a drop to background levels at 20 h and a gradual increase between days 2 and 6 of differentiation. The two lipin isoforms also exhibit differences in subcellular localization. Lipin-α is predominantly nuclear, whereas lipin-β is primarily located in the cytoplasm of 3T3-L1 adipocytes, suggesting distinct cellular functions. Using primary mouse embryonic fibroblasts expressing either lipin-α or lipin-β, we demonstrate functional differences between the two isoforms. Whereas lipin-α is required for adipocyte differentiation, the predominant effect of lipin-β expression is the induction of lipogenic genes. In vivo, overexpression of lipin-β specifically in mature adipocytes leads to elevated expression of lipogenic genes and adipocyte hypertrophy, confirming a role of lipin-β in the regulation of lipogenesis. In conclusion, our data suggest that the two lipin isoforms have distinct, but complementary, functions in adipogenesis, with lipin-α playing a primary role in differentiation and lipin-β being predominantly involved in lipogenesis.

Heterozygosity for Lmna deficiency eliminates the progeria-like phenotypes in Zmpste24-deficient mice

Zmpste24 is a metalloproteinase required for the processing of prelamin A to lamin A, a structural component of the nuclear lamina. Zmpste24 deficiency results in the accumulation of prelamin A within cells, a complete loss of mature lamin A, and misshapen nuclear envelopes. Zmpste24-deficient (Zmpste24–/–) mice exhibit retarded growth, alopecia, micrognathia, dental abnormalities, osteolytic lesions in bones, and osteoporosis, which are phenotypes shared with Hutchinson–Gilford progeria syndrome, a human disease caused by the synthesis of a mutant prelamin A that cannot undergo processing to lamin A. Zmpste24–/– mice also develop muscle weakness. We hypothesized that prelamin A might be toxic and that its accumulation in Zmpste24–/– mice is responsible for all of the disease phenotypes. We further hypothesized that Zmpste24–/– mice with half-normal levels of prelamin A (Zmpste24–/– mice with one Lmna knockout allele) would be subjected to less toxicity and be protected from disease. Thus, we bred and analyzed Zmpste24–/–Lmna+/– mice. As expected, prelamin A levels in Zmpste24–/–Lmna+/– cells were significantly reduced. Zmpste24–/–Lmna+/– mice were entirely normal, lacking all disease phenotypes, and misshapen nuclei were less frequent in Zmpste24–/–Lmna+/– cells than in Zmpste24–/– cells. These data suggest that prelamin A is toxic and that reducing its levels by as little as 50% provides striking protection from disease.

Cholesterol and Cholate Components of an Atherogenic Diet Induce Distinct Stages of Hepatic Inflammatory Gene Expression

Atherosclerosis in inbred mouse strains has been widely studied by using an atherogenic (Ath) diet containing cholesterol, cholic acid, and fat, but the effect of these components on gene expression has not been systematically examined. We employed DNA microarrays to interrogate gene expression levels in liver of C57BL/6J mice fed the following five diets: mouse chow, the Ath diet, or modified versions of the Ath diet in which either cholesterol, cholate, or fat were omitted. Dietary cholesterol and cholate produced discrete gene expression patterns. Cholesterol was required for induction of genes involved in acute inflammation, including three genes of the serum amyloid A family, three major histocompatibility class II antigen genes, and various cytokine-related genes. In contrast, cholate induced expression of genes involved in extracellular matrix deposition in hepatic fibrosis, including five collagen family members, collagen-interacting proteins, and connective tissue growth factor. The gene expression findings were confirmed by biochemical measurements showing that cholesterol was required for elevation of circulating serum amyloid A, and cholate was required for accumulation of collagen in the liver. The possibility that these gene expression changes are relevant to atherogenesis in C57BL/6J mice was supported by the observation that the closely related, yet atherosclerosis-resistant, C57BL/6ByJ strain was largely resistant to dietary induction of the inflammatory and fibrotic response genes. These results establish that cholesterol and cholate components of the Ath diet have distinct proatherogenic effects on gene expression and suggest a strategy to study the contribution of acute inflammatory response and fibrogenesis independently through dietary manipulation.