TTC39B destabilizes retinoblastoma protein promoting hepatic lipogenesis in a sex-specific fashion.

BACKGROUND & AIMS: Molecular mechanisms underlying the different susceptibility of men and women to non-alcoholic fatty liver disease (NAFLD) are poorly understood. The TTC39B locus encodes a scaffolding protein, associates with gynecological disorders and its deletion protects mice from diet-induced steatohepatitis. This study aimed to elucidate the molecular mechanisms linking TTC39B (T39) to the expression of lipogenic genes and to explore sex-specific effects. METHODS: Co-expression in HEK293A cells validated the novel T39/pRb interaction predicted by a protein-protein interaction algorithm. T39 was knocked down using an antisense oligonucleotide (ASO) in mice with dietary NAFLD and a genetic deficiency of pRb or its downstream effector E2F1, as well as in primary human hepatocytes. RESULTS: T39 interacts with pRb via its C-terminal TPR domain and promotes its proteasomal degradation. In female mice, T39 deficiency reduces the mRNA of lipogenic genes, especially Pnpla3, in a pRb- and E2F1-dependent manner. In contrast, in male mice, T39 deficiency results in a much smaller reduction in lipogenic gene expression that is independent of pRb/E2F1. T39 also interacts with VAPB via an N-terminal FFAT motif and stabilizes the interaction of VAPB with SCAP. Ovariectomy abolishes the effect of T39 knockdown on the hepatic pRb/E2F1/Pnpla3 axis. In both sexes T39 knockdown reduces SCAP independently of pRb. In primary human hepatocytes, T39 knockdown reduces expression of PNPLA3 and other lipogenic genes in women but not men. CONCLUSIONS: We have uncovered a conserved sexual dimorphism in the regulation of hepatic lipogenic genes, with effects of T39 mediated through pRb/E2F1 in females and VAPB/SCAP in both sexes. T39 inhibition could be a novel strategy to downregulate PNPLA3 and treat NAFLD in women. LAY SUMMARY: In females, the protein TTC39B degrades a tumor suppressor in the liver to promote the synthesis of new fat and the expression of a major genetic risk factor for non-alcoholic fatty liver disease. TTC39B is a potential therapeutic target for non-alcoholic fatty liver disease, especially in women.

Antisense oligonucleotide treatment produces a type I interferon response that protects against diet-induced obesity.

OBJECTIVE: In mouse models, deficiency of TTC39B (T39) decreases hepatic lipogenic gene expression and protects against diet-induced steatohepatitis. While assessing the therapeutic potential of antisense oligonucleotides (ASOs) targeting T39, we discovered an unexpected weight loss phenotype. The objective of this study was to determine the mechanism of the resistance to diet-induced obesity. METHODS: To assess therapeutic potential, we used antisense oligonucleotides (ASO) to knock down T39 expression in a Western or high-fat, high-cholesterol, high-sucrose-diet-fed Ldlr-/- or wild-type mice. RESULTS: T39 ASO treatment led to decreased hepatic lipogenic gene expression and decreased hepatic triglycerides. Unexpectedly, T39 ASO treatment protected against diet-induced obesity. The reduced weight gain was seen with two different ASOs that decreased T39 mRNA in adipose tissue macrophages (ATMs), but not with a liver-targeted GalNac-ASO. Mice treated with the T39 ASO displayed increased browning of gonadal white adipose tissue (gWAT) and evidence of increased lipolysis. However, T39 knockout mice displayed a similar weight loss response when treated with T39 ASO, indicating an off-target effect. RNA-seq analysis of gWAT showed a widespread increase in type I interferon (IFN)-responsive genes, and knockout of the IFN receptor abolished the weight loss phenotype induced by the T39 ASO. Some human T39 ASOs and ASOs with different modifications targeting LDLR also induced a type I IFN response in THP1 macrophages. CONCLUSION: Our data suggest that extrahepatic targeting of T39 by ASOs in ATMs produced an off-target type 1 IFN response, leading to activation of lipolysis, browning of WAT, and weight loss. While our findings suggest that ASOs may induce off-target type 1 IFN response more commonly than previously thought, they also suggest that therapeutic induction of type 1 IFN selectively in ATMs could potentially represent a novel approach to the treatment of obesity.

Calcitriol Accelerates Vascular Calcification Irrespective of Vitamin K Status in a Rat Model of Chronic Kidney Disease with Hyperphosphatemia and Secondary Hyperparathyroidism.

Patients with chronic kidney disease (CKD) have a markedly increased risk for developing cardiovascular disease. Nontraditional risk factors, such as increased phosphate retention, increased serum fibroblast growth factor 23 (FGF-23), and deficiencies in vitamins D and K metabolism, likely play key roles in the development of vascular calcification during CKD progression. Calcitriol [1,25-(OH)2-D3] is a key transcriptional regulator of matrix Gla protein, a vitamin K-dependent protein that inhibits vascular calcification. We hypothesized that calcitriol treatment would inhibit the development of vascular calcification and this inhibition would be dependent on vitamin K status in a rat model of CKD. Rats were treated with dietary adenine (0.25%) to induce CKD, with either 0, 20, or 80 ng/kg of calcitriol with low or high dietary vitamin K1 (0.2 or 100 mg/kg) for 7 weeks. Calcitriol at both lower (20 ng/kg) and moderate (80 ng/kg) doses increased the severity of vascular calcification, and contrary to our hypothesis this was not significantly improved by high dietary vitamin K1. Calcitriol had a dose-dependent effect on: 1) lowering serum parathyroid hormone, 2) increasing serum calcium, and 3) increasing serum FGF-23. Calcitriol treatment significantly increased aortic expression of the calcification genes Runx2 and Pit-1 These data also implicate impaired vitamin D catabolism in CKD, which may contribute to the development of calcitriol toxicity and increased vascular calcification. The present findings demonstrate that in an adenine-induced rat model of CKD calcitriol treatment at doses as low as 20 ng/kg can increase the severity of vascular calcification regardless of vitamin K status.

Vitamin K Metabolism in a Rat Model of Chronic Kidney Disease.

BACKGROUND: Patients with chronic kidney disease (CKD) have very high levels of uncarboxylated, inactive, extra-hepatic vitamin K-dependent proteins measured in circulation, putting them at risk for complications of vitamin K deficiency. The major form of vitamin K found in the liver is phylloquinone (K1). Menaquinone-4 (MK-4) is the form of vitamin K that is preferentially found in extra-hepatic tissues. METHODS: In the present study, we assessed tissue concentrations of K1 and MK-4 and the expression of vitamin K-related genes in a rat model of adenine-induced CKD. RESULTS: It was found that rats with both mild and severe CKD had significantly lower amounts of K1 measured in liver, spleen and heart and higher levels of MK-4 measured in kidney cortex and medulla. All animals treated with high dietary K1 had an increase in tissue levels of both K1 and MK-4; however, the relative increase in K1 differed suggesting that the conversion of K1 to MK-4 may be a regulated/limiting process in some tissues. There was a decrease in the thoracic aorta expression of vitamin K recycling (Vkor) and utilization (Ggcx) enzymes, and a decrease in the kidney level of vitamin K1 to MK-4 bioconversion enzyme Ubiad1 in CKD. CONCLUSION: Taken together, these findings suggest that CKD impacts vitamin K metabolism, and this occurs early in the disease course. Our findings that vitamin K metabolism is altered in the presence of CKD provides further support that sub-clinical vitamin K deficiency may represent a modifiable risk factor for vascular and bone health in this population.

Magnesium Modifies the Impact of Calcitriol Treatment on Vascular Calcification in Experimental Chronic Kidney Disease.

Chronic kidney disease (CKD) patients are commonly treated with vitamin D analogs, such as calcitriol. Recent epidemiologic evidence revealed a significant interaction between vitamin D and magnesium, since an inverse relationship between vitamin D levels and mortality mainly occurs in patients with a high magnesium intake. The aim of the study was to assess the mechanisms involved by determining whether magnesium alone or combined with calcitriol treatments differentially impacts vascular calcification (VC) in male Sprague-Dawley rats with adenine-induced CKD. Treatment with moderate doses of calcitriol (80 µg/kg) suppressed parathyroid hormone to near or slightly below control levels. Given alone, this dose of calcitriol increased the prevalence of VC; however, when magnesium was given in combination, the severity of calcification was attenuated in the abdominal aorta (51% reduction), iliac (44%), and carotid arteries (46%) compared with CKD controls. The decreases in vascular calcium content were associated with a 20-50% increase in vascular magnesium. Calcitriol treatment alone significantly decreased TRPM7 protein (↓ to ∼11%), whereas the combination treatment increased both mRNA (1.7×) and protein (6.8×) expression compared with calcitriol alone. In summary, calcitriol increased VC in certain conditions, but magnesium prevented the reduction in TRPM7 and reduced the severity of VC, thereby increasing the bioavailable magnesium in the vascular microenvironment. These findings suggest that modifying the adverse effect profile of calcitriol with magnesium may be a plausible approach to benefiting the increasing number of CKD patients being prescribed calcitriol.

Inhibiting the progression of arterial calcification with vitamin K in HemoDialysis patients (iPACK-HD) trial: rationale and study design for a randomized trial of vitamin K in patients with end stage kidney disease.

BACKGROUND: Cardiovascular disease, which is due in part to progressive vascular calcification, is the leading cause of death among patients with end stage kidney disease (ESKD) on dialysis. A role for vitamin K in the prevention of vascular calcification is plausible based on the presence of vitamin K dependent proteins in vascular tissue, including matrix gla protein (MGP). Evidence from animal models and observational studies support a role for vitamin K in the prevention of vascular calcification. A large-scale study is needed to investigate the effect of vitamin K supplementation on the progression of vascular calcification in patients with ESKD, a group at risk for sub-clinical vitamin K deficiency. METHODS/DESIGN: We plan a prospective, randomized, double-blind, multicenter controlled trial of incident ESKD patients on hemodialysis in centers within North America. Eligible subjects with a baseline coronary artery calcium score of greater than or equal to 30 Agatston Units, will be randomly assigned to either the treatment group (10 mg of phylloquinone three times per week) or to the control group (placebo administration three times per week). The primary endpoint is the progression of coronary artery calcification defined as a greater than 15% increase in CAC score over baseline after 12 months. DISCUSSION: Vitamin K supplementation is a simple, safe and cost-effective nutritional strategy that can easily be integrated into patient care. If vitamin K reduces the progression of coronary artery calcification it may lead to decreased morbidity and mortality in men and women with ESKD. TRIAL REGISTRATION: NCT 01528800.

CONTEXTE: La maladie cardiovasculaire, qui est partiellement attribuable à la calcification vasculaire progressive, est la cause principale de décès chez les patients atteints d'insuffisance rénale terminale (IRT) en hémodialyse. La vitamine K pourrait jouer un rôle dans la prévention de la calcification vasculaire, en raison de la présence de protéines dépendantes à la vitamine K dans les tissus vasculaires, dont l'ostéocalcine. Le modèle animal, de même que des études d'observation, témoignent du rôle de la vitamine K dans la prévention de la calcification vasculaire. Une étude réalisée à grande échelle serait nécessaire afin d'étudier l'effet de la supplémentation de vitamine K sur la progression de la calcification vasculaire chez les patients atteints d'IRT, un groupe à risque pour les carences infracliniques en vitamine K. MÉTHODE/TYPE D'ÉTUDE: Nous prévoyons effectuer un essai clinique aléatoire, à double insu et multicentrique auprès de patients atteints d'IRT, traités en hémodialyse hospitalière en Amérique du Nord. On affectera au hasard les sujets admissibles qui présentent dans l'artère coronaire un taux de calcium supérieur ou égal à 30 unités d'Agatston, soit au groupe auquel on administre un traitement (10 mg de phylloquinone trois fois par semaine), soit au groupe témoin (administration du placebo trois fois par semaine). Le critère d'évaluation principal est la progression de la calcification de l'artère coronaire, définie comme une augmentation supérieure à 15% (résultat CAC) par rapport au point de référence, au bout de douze mois. DISCUSSION: La supplémentation de vitamine K est une stratégie nutritionnelle à la fois simple, sécuritaire et rentable, qui peut être aisément intégrée aux soins aux patients. S'il s'avère que la vitamine K réduit la progression de la calcification de l'artère coronaire, il pourrait y avoir une diminution de la morbidité et de la mortalité chez les hommes et les femmes atteints d'IRT.

Calcification of the internal pudendal artery and development of erectile dysfunction in adenine-induced chronic kidney disease: a sentinel of systemic vascular changes.

INTRODUCTION: Chronic kidney disease (CKD), erectile dysfunction (ED), and cardiovascular disease share common vascular etiologies and risk factors. AIM: Using a rat model, this is the first study to characterize the consequences of CKD in the onset and development of ED associated with differential regional vascular calcification and circulatory changes. METHODS: Stable CKD was generated at 3 weeks in male Sprague-Dawley rats given dietary adenine and progressed until 7 weeks. Mineral content and morphometry were assessed in the internal pudendal arteries (IPAs), thoracic aorta, and carotid artery. Endothelial function was determined via changes in serum von Willebrand factor (VWF) and endothelium-dependent relaxation of the thoracic aorta. RESULTS: In severe CKD rats, calcium and phosphate content in all arteries increased, and pulse wave velocity was elevated. Distal IPA segments, in particular, were the first to calcify, but penile tissue per se did not. CKD rats had endothelial dysfunction, as indicated by a decrease in acetylcholine-mediated relaxation (∼40%) and an increase in serum VWF (∼40%), as well as increased lumen diameter (20%) of the distal IPA. Erectile function, assessed using a centrally acting dopaminergic agent, was significantly impaired by 7 weeks (∼40%). CONCLUSIONS: In CKD, the distal IPA appears to be more susceptible to vascular dysfunction and calcification. Additionally, the onset of ED may be an important sentinel of impending systemic vascular disease. To confirm this concept, future experimental and clinical studies will need to examine a range of vessel types and the use of supplementary methods to assess erectile function.

Vitamin K status in chronic kidney disease.

The purpose of this review is to summarize the research to date on vitamin K status in chronic kidney disease (CKD). This review includes a summary of the data available on vitamin K status in patients across the spectrum of CKD as well as the link between vitamin K deficiency in CKD and bone dynamics, including mineralization and demineralization, as well as ectopic mineralization. It also describes two current clinical trials that are underway evaluating vitamin K treatment in CKD patients. These data may inform future clinical practice in this population.

Dietary vitamin K and therapeutic warfarin alter the susceptibility to vascular calcification in experimental chronic kidney disease.

The leading cause of death in patients with chronic kidney disease (CKD) is cardiovascular disease, with vascular calcification being a key modifier of disease progression. A local regulator of vascular calcification is vitamin K. This γ-glutamyl carboxylase substrate is an essential cofactor in the activation of several extracellular matrix proteins that inhibit calcification. Warfarin, a common therapy in dialysis patients, inhibits the recycling of vitamin K and thereby decreases the inhibitory activity of these proteins. In this study, we sought to determine whether modifying vitamin K status, either by increasing dietary vitamin K intake or by antagonism with therapeutic doses of warfarin, could alter the development of vascular calcification in male Sprague-Dawley rats with adenine-induced CKD. Treatment of CKD rats with warfarin markedly increased pulse pressure and pulse wave velocity, as well as significantly increased calcium concentrations in the thoracic aorta (3-fold), abdominal aorta (8-fold), renal artery (4-fold), and carotid artery (20-fold). In contrast, treatment with high dietary vitamin K1 increased vitamin K tissue concentrations (10-300-fold) and blunted the development of vascular calcification. Thus, vitamin K has an important role in modifying mechanisms linked to the susceptibility of arteries to calcify in an experimental model of CKD.