Dysregulation of Lipid and Glucose Homeostasis in Hepatocyte-Specific SLC25A34 Knockout Mice.

Nonalcoholic fatty liver disease (NAFLD) is an epidemic affecting 30% of the US population. It is characterized by insulin resistance, and by defective lipid metabolism and mitochondrial dysfunction in the liver. SLC25A34 is a major repressive target of miR-122, a miR that has a central role in NAFLD and liver cancer. However, little is known about the function of SLC25A34. To investigate SLC25A34 in vitro, mitochondrial respiration and bioenergetics were examined using hepatocytes depleted of Slc25a34 or overexpressing Slc25a34. To test the function of SLC25A34 in vivo, a hepatocyte-specific knockout mouse was generated, and loss of SLC25A34 was assessed in mice maintained on a chow diet and a fast-food diet (FFD), a model for NAFLD. Hepatocytes depleted of Slc25a34 displayed increased mitochondrial biogenesis, lipid synthesis, and ADP/ATP ratio; Slc25a34 overexpression had the opposite effect. In the knockout model on chow diet, SLC25A34 loss modestly affected liver function (altered glucose metabolism was the most pronounced defect). RNA-sequencing revealed changes in metabolic processes, especially fatty acid metabolism. After 2 months on FFD, knockouts had a more severe phenotype, with increased lipid content and impaired glucose tolerance, which was attenuated after longer FFD feeding (6 months). This work thus presents a novel model for studying SLC25A34 in vivo in which SLC25A34 plays a role in mitochondrial respiration and bioenergetics during NAFLD.

Inflammation and Ectopic Fat Deposition in the Aging Murine Liver Is Influenced by CCR2.

Aging is associated with inflammation and metabolic syndrome, which manifests in the liver as nonalcoholic fatty liver disease (NAFLD). NAFLD can range in severity from steatosis to fibrotic steatohepatitis and is a major cause of hepatic morbidity. However, the pathogenesis of NAFLD in naturally aged animals is unclear. Herein, we performed a comprehensive study of lipid content and inflammatory signature of livers in 19-month-old aged female mice. These animals exhibited increased body and liver weight, hepatic triglycerides, and inflammatory gene expression compared with 3-month-old young controls. The aged mice also had a significant increase in F4/80+ hepatic macrophages, which coexpressed CD11b, suggesting a circulating monocyte origin. A global knockout of the receptor for monocyte chemoattractant protein (CCR2) prevented excess steatosis and inflammation in aging livers but did not reduce the number of CD11b+ macrophages, suggesting changes in macrophage accumulation precede or are independent from chemokine (C-C motif) ligand-CCR2 signaling in the development of age-related NAFLD. RNA sequencing further elucidated complex changes in inflammatory and metabolic gene expression in the aging liver. In conclusion, we report a previously unknown accumulation of CD11b+ macrophages in aged livers with robust inflammatory and metabolic transcriptomic changes. A better understanding of the hallmarks of aging in the liver will be crucial in the development of preventive measures and treatments for end-stage liver disease in elderly patients.

Effects of Proximal Tubule Shortening on Protein Excretion in a Lowe Syndrome Model.

BACKGROUND: Lowe syndrome (LS) is an X-linked recessive disorder caused by mutations in OCRL, which encodes the enzyme OCRL. Symptoms of LS include proximal tubule (PT) dysfunction typically characterized by low molecular weight proteinuria, renal tubular acidosis (RTA), aminoaciduria, and hypercalciuria. How mutant OCRL causes these symptoms isn't clear. METHODS: We examined the effect of deleting OCRL on endocytic traffic and cell division in newly created human PT CRISPR/Cas9 OCRL knockout cells, multiple PT cell lines treated with OCRL-targeting siRNA, and in orcl-mutant zebrafish. RESULTS: OCRL-depleted human cells proliferated more slowly and about 10% of them were multinucleated compared with fewer than 2% of matched control cells. Heterologous expression of wild-type, but not phosphatase-deficient, OCRL prevented the accumulation of multinucleated cells after acute knockdown of OCRL but could not rescue the phenotype in stably edited knockout cell lines. Mathematic modeling confirmed that reduced PT length can account for the urinary excretion profile in LS. Both ocrl mutant zebrafish and zebrafish injected with ocrl morpholino showed truncated expression of megalin along the pronephric kidney, consistent with a shortened S1 segment. CONCLUSIONS: Our data suggest a unifying model to explain how loss of OCRL results in tubular proteinuria as well as the other commonly observed renal manifestations of LS. We hypothesize that defective cell division during kidney development and/or repair compromises PT length and impairs kidney function in LS patients.

Polyploid Hepatocytes Facilitate Adaptation and Regeneration to Chronic Liver Injury.

The liver contains diploid and polyploid hepatocytes (tetraploid, octaploid, etc.), with polyploids comprising ≥90% of the hepatocyte population in adult mice. Polyploid hepatocytes form multipolar spindles in mitosis, which lead to chromosome gains/losses and random aneuploidy. The effect of aneuploidy on liver function is unclear, and the degree of liver aneuploidy is debated, with reports showing aneuploidy affects 5% to 60% of hepatocytes. To study relationships among liver polyploidy, aneuploidy, and adaptation, mice lacking E2f7 and E2f8 in the liver (LKO), which have a polyploidization defect, were used. Polyploids were reduced fourfold in LKO livers, and LKO hepatocytes remained predominantly diploid after extensive proliferation. Moreover, nearly all LKO hepatocytes were euploid compared with control hepatocytes, suggesting polyploid hepatocytes are required for production of aneuploid progeny. To determine whether reduced polyploidy impairs adaptation, LKO mice were bred onto a tyrosinemia background, a disease model whereby the liver can develop disease-resistant, regenerative nodules. Although tyrosinemic LKO mice were more susceptible to morbidities and death associated with tyrosinemia-induced liver failure, they developed regenerating nodules similar to control mice. Analyses revealed that nodules in the tyrosinemic livers were generated by aneuploidy and inactivating mutations. In summary, we identified new roles for polyploid hepatocytes and demonstrated that they are required for the formation of aneuploid progeny and can facilitate adaptation to chronic liver disease.

The Polyploid State Restricts Hepatocyte Proliferation and Liver Regeneration in Mice.

The liver contains a mixture of hepatocytes with diploid or polyploid (tetraploid, octaploid, etc.) nuclear content. Polyploid hepatocytes are commonly found in adult mammals, representing ~90% of the entire hepatic pool in rodents. The cellular and molecular mechanisms that regulate polyploidization have been well characterized; however, it is unclear whether diploid and polyploid hepatocytes function similarly in multiple contexts. Answering this question has been challenging because proliferating hepatocytes can increase or decrease ploidy, and animal models with healthy diploid-only livers have not been available. Mice lacking E2f7 and E2f8 in the liver (liver-specific E2f7/E2f8 knockout; LKO) were recently reported to have a polyploidization defect, but were otherwise healthy. Herein, livers from LKO mice were rigorously characterized, demonstrating a 20-fold increase in diploid hepatocytes and maintenance of the diploid state even after extensive proliferation. Livers from LKO mice maintained normal function, but became highly tumorigenic when challenged with tumor-promoting stimuli, suggesting that tumors in LKO mice were driven, at least in part, by diploid hepatocytes capable of rapid proliferation. Indeed, hepatocytes from LKO mice proliferate faster and out-compete control hepatocytes, especially in competitive repopulation studies. In addition, diploid or polyploid hepatocytes from wild-type (WT) mice were examined to eliminate potentially confounding effects associated with E2f7/E2f8 deficiency. WT diploid cells also showed a proliferative advantage, entering and progressing through the cell cycle faster than polyploid cells, both in vitro and during liver regeneration (LR). Diploid and polyploid hepatocytes responded similarly to hepatic mitogens, indicating that proliferation kinetics are unrelated to differential response to growth stimuli. Conclusion: Diploid hepatocytes proliferate faster than polyploids, suggesting that the polyploid state functions as a growth suppressor to restrict proliferation by the majority of hepatocytes.

Inhibition of Glycogen Synthase Kinase 3 Accelerated Liver Regeneration after Acetaminophen-Induced Hepatotoxicity in Mice.

Overdose of acetaminophen (APAP) is the leading cause of acute liver failure (ALF) in the United States. Timely initiation of compensatory liver regeneration after APAP hepatotoxicity is critical for final recovery, but the mechanisms of liver regeneration after APAP-induced ALF have not been extensively explored yet. Previous studies from our laboratory have demonstrated that activation of ß-catenin signaling after APAP overdose is associated with timely liver regeneration. Herein, we investigated the role of glycogen synthase kinase 3 (GSK3) in liver regeneration after APAP hepatotoxicity using a pharmacological inhibition strategy in mice. Treatment with specific GSK3 inhibitor (L803-mts), starting from 4 hours after 600 mg/kg dose of APAP, resulted in early initiation of liver regeneration in a dose-dependent manner, without modifying the peak regenerative response. Acceleration of liver regeneration was not secondary to alteration of APAP-induced hepatotoxicity, which remained unchanged after GSK3 inhibition. Early cell cycle initiation in hepatocytes after GSK3 inhibition was because of rapid induction of cyclin D1 and phosphorylation of retinoblastoma protein. This was associated with increased activation of ß-catenin signaling after GSK3 inhibition. Taken together, our study has revealed a novel role of GSK3 in liver regeneration after APAP overdose and identified GSK3 as a potential therapeutic target to improve liver regeneration after APAP-induced ALF.

Oxaloacetate activates brain mitochondrial biogenesis, enhances the insulin pathway, reduces inflammation and stimulates neurogenesis.

Brain bioenergetic function declines in some neurodegenerative diseases, this may influence other pathologies and administering bioenergetic intermediates could have therapeutic value. To test how one intermediate, oxaloacetate (OAA) affects brain bioenergetics, insulin signaling, inflammation and neurogenesis, we administered intraperitoneal OAA, 1-2 g/kg once per day for 1-2 weeks, to C57Bl/6 mice. OAA altered levels, distributions or post-translational modifications of mRNA and proteins (proliferator-activated receptor-gamma coactivator 1α, PGC1 related co-activator, nuclear respiratory factor 1, transcription factor A of the mitochondria, cytochrome oxidase subunit 4 isoform 1, cAMP-response element binding, p38 MAPK and adenosine monophosphate-activated protein kinase) in ways that should promote mitochondrial biogenesis. OAA increased Akt, mammalian target of rapamycin and P70S6K phosphorylation. OAA lowered nuclear factor κB nucleus-to-cytoplasm ratios and CCL11 mRNA. Hippocampal vascular endothelial growth factor mRNA, doublecortin mRNA, doublecortin protein, doublecortin-positive neuron counts and neurite length increased in OAA-treated mice. (1)H-MRS showed OAA increased brain lactate, GABA and glutathione thereby demonstrating metabolic changes are detectable in vivo. In mice, OAA promotes brain mitochondrial biogenesis, activates the insulin signaling pathway, reduces neuroinflammation and activates hippocampal neurogenesis.

Bile acids promote diethylnitrosamine-induced hepatocellular carcinoma via increased inflammatory signaling.

Hepatocellular carcinoma (HCC) is the most common hepatic malignancy and the third leading cause of cancer related deaths. Previous studies have implicated bile acids in pathogenesis of HCC, but the mechanisms are not known. We investigated the mechanisms of HCC tumor promotion by bile acids the diethylnitrosamine (DEN)-initiation-cholic acid (CA)-induced tumor promotion protocol in mice. The data show that 0.2% CA treatment resulted in threefold increase in number and size of DEN-induced liver tumors. All tumors observed in DEN-treated mice were well-differentiated HCCs. The HCCs observed in DEN-treated CA-fed mice exhibited extensive CD3-, CD20-, and CD45-positive inflammatory cell aggregates. Microarray-based global gene expression studies combined with Ingenuity Pathway Analysis revealed significant activation of NF-κB and Nanog in the DEN-treated 0.2% CA-fed livers. Further studies showed significantly higher TNF-α and IL-1ß mRNA, a marked increase in total and phosphorylated-p65 and phosphorylated IκBα (degradation form) in livers of DEN-treated 0.2% CA-fed mice. Treatment of primary mouse hepatocytes with various bile acids showed significant induction of stemness genes including Nanog, KLF4, Sox2, and Oct4. Quantification of total and 20 specific bile acids in liver, and serum revealed a tumor-associated bile acid signature. Finally, quantification of total serum bile acids in normal, cirrhotic, and HCC human samples revealed increased bile acids in serum of cirrhotic and HCC patients. Taken together, these data indicate that bile acids are mechanistically involved pathogenesis of HCC and may promote HCC formation via activation of inflammatory signaling.

Bioenergetic flux, mitochondrial mass and mitochondrial morphology dynamics in AD and MCI cybrid cell lines.

Bioenergetic dysfunction occurs in Alzheimer's disease (AD) and mild cognitive impairment (MCI), a clinical syndrome that frequently precedes symptomatic AD. In this study, we modeled AD and MCI bioenergetic dysfunction by transferring mitochondria from MCI, AD and control subject platelets to mtDNA-depleted SH-SY5Y cells. Bioenergetic fluxes and bioenergetics-related infrastructures were characterized in the resulting cytoplasmic hybrid (cybrid) cell lines. Relative to control cybrids, AD and MCI cybrids showed changes in oxygen consumption, respiratory coupling and glucose utilization. AD and MCI cybrids had higher ADP/ATP and lower NAD+/NADH ratios. AD and MCI cybrids exhibited differences in proteins that monitor, respond to or regulate cell bioenergetic fluxes including HIF1α, PGC1α, SIRT1, AMPK, p38 MAPK and mTOR. Several endpoints suggested mitochondrial mass increased in the AD cybrid group and probably to a lesser extent in the MCI cybrid group, and that the mitochondrial fission-fusion balance shifted towards increased fission in the AD and MCI cybrids. As many of the changes we observed in AD and MCI cybrid models are also seen in AD subject brains, we conclude reduced bioenergetic function is present during very early AD, is not brain-limited and induces protean retrograde responses that likely have both adaptive and mal-adaptive consequences.

Regulation of mitochondrial function and cellular energy metabolism by protein kinase C-λ/ι: a novel mode of balancing pluripotency.

Pluripotent stem cells (PSCs) contain functionally immature mitochondria and rely upon high rates of glycolysis for their energy requirements. Thus, altered mitochondrial function and promotion of aerobic glycolysis are key to maintain and induce pluripotency. However, signaling mechanisms that regulate mitochondrial function and reprogram metabolic preferences in self-renewing versus differentiated PSC populations are poorly understood. Here, using murine embryonic stem cells (ESCs) as a model system, we demonstrate that atypical protein kinase C isoform, PKC lambda/iota (PKCλ/ι), is a key regulator of mitochondrial function in ESCs. Depletion of PKCλ/ι in ESCs maintains their pluripotent state as evident from germline offsprings. Interestingly, loss of PKCλ/ι in ESCs leads to impairment in mitochondrial maturation, organization, and a metabolic shift toward glycolysis under differentiating condition. Our mechanistic analyses indicate that a PKCλ/ι-hypoxia-inducible factor 1α-PGC1α axis regulates mitochondrial respiration and balances pluripotency in ESCs. We propose that PKCλ/ι could be a crucial regulator of mitochondrial function and energy metabolism in stem cells and other cellular contexts.

Extracellular Mitochondria and Mitochondrial Components Act as Damage-Associated Molecular Pattern Molecules in the Mouse Brain.

Mitochondria and mitochondrial debris are found in the brain's extracellular space, and extracellular mitochondrial components can act as damage associated molecular pattern (DAMP) molecules. To characterize the effects of potential mitochondrial DAMP molecules on neuroinflammation, we injected either isolated mitochondria or mitochondrial DNA (mtDNA) into hippocampi of C57BL/6 mice and seven days later measured markers of inflammation. Brains injected with whole mitochondria showed increased Tnfα and decreased Trem2 mRNA, increased GFAP protein, and increased NFκB phosphorylation. Some of these effects were also observed in brains injected with mtDNA (decreased Trem2 mRNA, increased GFAP protein, and increased NFκB phosphorylation), and mtDNA injection also caused several unique changes including increased CSF1R protein and AKT phosphorylation. To further establish the potential relevance of this response to Alzheimer's disease (AD), a brain disorder characterized by neurodegeneration, mitochondrial dysfunction, and neuroinflammation we also measured App mRNA, APP protein, and Aß1-42 levels. We found mitochondria (but not mtDNA) injections increased these parameters. Our data show that in the mouse brain extracellular mitochondria and its components can induce neuroinflammation, extracellular mtDNA or mtDNA-associated proteins can contribute to this effect, and mitochondria derived-DAMP molecules can influence AD-associated biomarkers.

Effect of cholinergic signaling on neuronal cell bioenergetics.

Alzheimer's disease (AD) patients have reduced brain acetylcholine and reversing this deficit yields clinical benefits. In this study we explored how increased cholinergic tone impacts cell bioenergetics, which are also perturbed in AD. We treated SH-SY5Y neuroblastoma cells with carbachol, a cholinergic agonist, and tested for bioenergetic flux and bioenergetic infrastructure changes. Carbachol rapidly increased both oxidative phosphorylation and glycolysis fluxes. ATP levels rose slightly, as did cell energy demand, and AMPK phosphorylation occurred. At least some of these effects depended on muscarinic receptor activation, ER calcium release, and ER calcium re-uptake. Our data show that increasing cholinergic signaling enhances cell bioenergetics, and reveal mechanisms that mediate this effect. Phenomena we observed could potentially explain why cholinesterase inhibitor therapy increases AD brain glucose utilization and N-acetyl aspartate levels. The question of whether cholinesterase inhibitors have a disease modifying effect in AD has long been debated; our data suggest a theoretical mechanism through which such an effect could potentially arise.