Regulation of Metabolism by Hepatic OXPHOS: A Dissertation

Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent issue in the modern world, predisposing patients to serious pathology such as cirrhosis and hepatocellular carcinoma. Mitochondrial dysfunction, and in particular, diminished hepatic oxidative phosphorylation (OXPHOS) capacity, have been observed in NAFLD livers, which may participate in NAFLD pathogenesis. To examine the role of OXPHOS in NAFLD, we generated a model of enhanced hepatic OXPHOS using mice with liver-specific transgenic expression of LRPPRC, a protein which activates mitochondrial transcription and augments OXPHOS capacity. When challenged with high-fat feeding, mice with enhanced hepatic OXPHOS were protected from the development of liver steatosis and inflammation, critical components in the pathogenesis of NAFLD. This protection corresponded to increased liver and whole-body insulin sensitivity. Moreover, mice with enhanced hepatic OXPHOS have increased availability of oxidized NAD+, which promotes complete fatty acid oxidation in hepatocytes. Interestingly, mice with enhanced hepatic OXPHOS were also protected from obesogenic effects of long-term high-fat feeding. Consistent with this, enhanced hepatic OXPHOS increased energy expenditure and adipose tissue oxidative gene expression, suggesting a communication between the liver and adipose tissue to promote thermogenesis. Examination of pro-thermogenic molecules revealed altered bile acid composition in livers and serum of LRPPRC transgenic mice. These mice had increased expression of bile acid synthetic enzymes, genes which are induced by NAD+ dependent deacetylase SIRT1 activation of the transcriptional co-regulator PGC-1a. These findings suggest that enhanced hepatic OXPHOS transcriptionally regulates bile acid synthesis and dictates whole-body energy expenditure, culminating in protection from obesity

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders. In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via beta-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying beta-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of beta-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates

OXPHOS-Mediated Induction of NAD+ Promotes Complete Oxidation of Fatty Acids and Interdicts Non-Alcoholic Fatty Liver Disease

OXPHOS is believed to play an important role in non-alcoholic fatty liver disease (NAFLD), however, precise mechanisms whereby OXPHOS influences lipid homeostasis are incompletely understood. We previously reported that ectopic expression of LRPPRC, a protein that increases cristae density and OXPHOS, promoted fatty acid oxidation in cultured primary hepatocytes. To determine the biological significance of that observation and define underlying mechanisms, we have ectopically expressed LRPPRC in mouse liver in the setting of NAFLD. Interestingly, ectopic expression of LRPPRC in mouse liver completely interdicted NAFLD, including inflammation. Consistent with mitigation of NAFLD, two markers of hepatic insulin resistance-ROS and PKCepsilon activity-were both modestly reduced. As reported by others, improvement of NAFLD was associated with improved whole-body insulin sensitivity. Regarding hepatic lipid homeostasis, the ratio of NAD+ to NADH was dramatically increased in mouse liver replete with LRPPRC. Pharmacological activators and inhibitors of the cellular respiration respectively increased and decreased the [NAD+]/[NADH] ratio, indicating respiration-mediated control of the [NAD+]/[NADH] ratio. Supporting a prominent role for NAD+, increasing the concentration of NAD+ stimulated complete oxidation of fatty acids. Importantly, NAD+ rescued impaired fatty acid oxidation in hepatocytes deficient for either OXPHOS or SIRT3. These data are consistent with a model whereby augmented hepatic OXPHOS increases NAD+, which in turn promotes complete oxidation of fatty acids and protects against NAFLD

Mitochondrial retrograde signaling connects respiratory capacity to thermogenic gene expression

Mitochondrial respiration plays a crucial role in determining the metabolic state of brown adipose tissue (BAT), due to its direct roles in thermogenesis, as well as through additional mechanisms. Here, we show that respiration-dependent retrograde signaling from mitochondria to nucleus contributes to genetic and metabolic reprogramming of BAT. In mouse BAT, ablation of LRPPRC (LRP130), a potent regulator of mitochondrial transcription and respiratory capacity, triggers down-regulation of thermogenic genes, promoting a storage phenotype in BAT. This retrograde regulation functions by inhibiting the recruitment of PPARgamma to the regulatory elements of thermogenic genes. Reducing cytosolic Ca2+ reverses the attenuation of thermogenic genes in brown adipocytes with impaired respiratory capacity, while induction of cytosolic Ca2+ is sufficient to attenuate thermogenic gene expression, indicating that cytosolic Ca2+ mediates mitochondria-nucleus crosstalk. Our findings suggest respiratory capacity governs thermogenic gene expression and BAT function via mitochondria-nucleus communication, which in turn leads to either a thermogenic or storage mode

Nutrient sensing by the mitochondrial transcription machinery dictates oxidative phosphorylation

Sirtuin 3 (SIRT3), an important regulator of energy metabolism and lipid oxidation, is induced in fasted liver mitochondria and implicated in metabolic syndrome. In fasted liver, SIRT3-mediated increases in substrate flux depend on oxidative phosphorylation (OXPHOS), but precisely how OXPHOS meets the challenge of increased substrate oxidation in fasted liver remains unclear. Here, we show that liver mitochondria in fasting mice adapt to the demand of increased substrate oxidation by increasing their OXPHOS efficiency. In response to cAMP signaling, SIRT3 deacetylated and activated leucine-rich protein 130 (LRP130; official symbol, LRPPRC), promoting a mitochondrial transcriptional program that enhanced hepatic OXPHOS. Using mass spectrometry, we identified SIRT3-regulated lysine residues in LRP130 that generated a lysine-to-arginine (KR) mutant of LRP130 that mimics deacetylated protein. Compared with wild-type LRP130 protein, expression of the KR mutant increased mitochondrial transcription and OXPHOS in vitro. Indeed, even when SIRT3 activity was abolished, activation of mitochondrial transcription and OXPHOS by the KR mutant remained robust, further highlighting the contribution of LRP130 deacetylation to increased OXPHOS in fasted liver. These data establish a link between nutrient sensing and mitochondrial transcription that regulates OXPHOS in fasted liver and may explain how fasted liver adapts to increased substrate oxidation

Direct Recruitment of Polycomb Repressive Complex 1 to Chromatin by Core Binding Transcription Factors

Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFβ transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate significant chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFβ and functional regulation of a considerable fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFβ deficiencies generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via PRC2-independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.Stem Cell and Regenerative Biolog

beta3-Adrenergic receptor stimulation induces E-selectin-mediated adipose tissue inflammation

Inflammation induced by wound healing or infection activates local vascular endothelial cells to mediate leukocyte rolling, adhesion, and extravasation by up-regulation of leukocyte adhesion molecules such as E-selectin and P-selectin. Obesity-associated adipose tissue inflammation has been suggested to cause insulin resistance, but weight loss and lipolysis also promote adipose tissue immune responses. While leukocyte-endothelial interactions are required for obesity-induced inflammation of adipose tissue, it is not known whether lipolysis-induced inflammation requires activation of endothelial cells. Here, we show that beta(3)-adrenergic receptor stimulation by CL 316,243 promotes adipose tissue neutrophil infiltration in wild type and P-selectin-null mice but not in E-selectin-null mice. Increased expression of adipose tissue cytokines IL-1beta, CCL2, and TNF-alpha in response to CL 316,243 administration is also dependent upon E-selectin but not P-selectin. In contrast, fasting increases adipose-resident macrophages but not neutrophils, and does not activate adipose-resident endothelium. Thus, two models of lipolysis-induced inflammation induce distinct immune cell populations within adipose tissue and exhibit distinct dependences on endothelial activation. Importantly, our results indicate that beta(3)-adrenergic stimulation acts through up-regulation of E-selectin in adipose tissue endothelial cells to induce neutrophil infiltration

Augmented hepatic OXPHOS interdicts NAFLD.

<p>(A) Representative histological sections of livers from wild-type littermate control mice (WT) and LRPPRC double hemizygous (Tg/Tg) mice. (B) Pathologic classification of inflammation in livers of the same mice. (C) Expression of inflammatory genes in livers of WT and Tg/Tg mice fed a high-fat diet for 12 weeks. (D) Pathologic classification of steatosis in high-fat fed WT and Tg/Tg mouse livers. (E) Biochemical determination of triglyceride content in the same samples. (F) Determination of <i>Fsp27</i> expression in high-fat fed WT and Tg/Tg livers. (G) Serum triglyceride content in the same mice. Expression of genes involved in (H) lipid export, (I) lipid uptake, (J) Lipogenesis, and (K) fatty acid β-oxidation in high-fat fed WT and Tg/Tg mouse livers. For all experiments, n = 9 or 10. Data are mean ± SEM *p<0.05, **p<0.01, ***p<0.001 vs. WT by chi-squared analysis (B, D), 2-way ANOVA with Bonferroni’s post-test (C), or by two-tailed, unpaired Student’s t-test (D-K).</p

Hepatic OXPHOS enhances whole body insulin sensitivity.

<p>(A) Glucose and (B) insulin tolerance tests and area-under-the-curve (insets) and (C) weights in wild-type littermate control mice (WT), LRPPRC single hemizygous (Tg/0), and double hemizygous (Tg/Tg) mice fed a high-fat diet for 12 weeks, n = 10. (D) Hourly activity, (E) daily food intake, and (F) diurnal energy expenditure (7AM—7PM) in WT and Tg/Tg mice fed a high-fat diet > 20 weeks n = 3–6. (G) ER stress and (H) antioxidant gene expression in high-fat fed WT and Tg/Tg mouse liver, n = 9 or 10. (I) Mitochondrial superoxide production and (J) mitochondrial membrane potential (ΔΨ_m) in HepG2 cells ectopically expressing LacZ (control) or LRPPRC n = 24. (K) Immunoblot and quantification of phospho-AKT in cultured primary hepatocytes from chow-fed WT and Tg/Tg mice treated with 25nM insulin, n = 3. (L) Immunoblot and (M) quantification of membrane or (N) membrane: cytosolic PKCε in high-fat fed WT and Tg/Tg mouse livers, n = 5 or 6. Data are mean ± SEM, *p<0.05, **p<0.01, ***p<0.001 vs. WT by two-way ANOVA (A, B), two-tailed unpaired Student’s t-test (insets, F, H, J, K), mixed model with Bonferroni correction (C) or one-tailed unpaired Student’s t-test (M, N).</p

LRPPRC promotes mitochondrially encoded gene expression in liver.

<p>(A) Protein expression and (B) quantification in livers of wild-type littermate controls (WT), hemizygous (Tg/0), and double hemizygous (Tg/Tg) LRPPRC transgenic mice. (C) Protein expression in heart, spleen, brown adipose tissue (BAT) and liver in WT and Tg/Tg mice. (D) Protein expression in WT and Tg/Tg cultured primary hepatocytes. (E) Nuclear <i>Lrpprc</i>, mitochondrial polymerase (<i>Polrmt</i>), mitochondrial transcription factor A (<i>Tfam</i>), and mitochondrial transcription factor B2 (<i>Tfb2m</i>) and (F) mitochondrially encoded respiratory complex subunit gene expression in Tg/Tg and control livers. (G) Genetic determination of mitochondrially encoded DNA content in the samples. For all experiments, n = 3 or 4. Data are mean ± SEM * p<0.05, ** p<0.01, *** p<0.001 vs. WT by two-tailed unpaired Student’s t-test (B, E, G) or 2-way ANOVA with Bonferroni’s post-test (F).</p