A spatial map of hepatic mitochondria uncovers functional heterogeneity shaped by nutrient-sensing signaling.

In the liver, mitochondria are exposed to different concentrations of nutrients due to their spatial positioning across the periportal and pericentral axis. How the mitochondria sense and integrate these signals to respond and maintain homeostasis is not known. Here, we combine intravital microscopy, spatial proteomics, and functional assessment to investigate mitochondrial heterogeneity in the context of liver zonation. We find that periportal and pericentral mitochondria are morphologically and functionally distinct; beta-oxidation is elevated in periportal regions, while lipid synthesis is predominant in the pericentral mitochondria. In addition, comparative phosphoproteomics reveals spatially distinct patterns of mitochondrial composition and potential regulation via phosphorylation. Acute pharmacological modulation of nutrient sensing through AMPK and mTOR shifts mitochondrial phenotypes in the periportal and pericentral regions, linking nutrient gradients across the lobule and mitochondrial heterogeneity. This study highlights the role of protein phosphorylation in mitochondrial structure, function, and overall homeostasis in hepatic metabolic zonation. These findings have important implications for liver physiology and disease.

A spatial map of hepatic mitochondria uncovers functional heterogeneity shaped by nutrient-sensing signaling.

In the liver, mitochondria are exposed to different concentrations of nutrients due to their spatial positioning across the periportal (PP) and pericentral (PC) axis. How these mitochondria sense and integrate these signals to respond and maintain homeostasis is not known. Here, we combined intravital microscopy, spatial proteomics, and functional assessment to investigate mitochondrial heterogeneity in the context of liver zonation. We found that PP and PC mitochondria are morphologically and functionally distinct; beta-oxidation was elevated in PP regions, while lipid synthesis was predominant in the PC mitochondria. In addition, comparative phosphoproteomics revealed spatially distinct patterns of mitochondrial composition and potential regulation via phosphorylation. Acute pharmacological modulation of nutrient sensing through AMPK and mTOR shifted mitochondrial phenotypes in the PP and PC regions, linking nutrient gradients across the lobule and mitochondrial heterogeneity. This study highlights the role of protein phosphorylation in mitochondrial structure, function, and overall homeostasis in hepatic metabolic zonation. These findings have important implications for liver physiology and disease.

Location matters: cellular heterogeneity in the hepatic lobule and hepatocellular carcinoma.

Tumor heterogeneity is a hallmark of cancer but a challenging problem to dissect mechanistically. Less recognized is that cells within normal tissues are also remarkably diverse. Hepatocytes are a great example because their spatial positioning and the local microenvironment govern their genetic heterogeneity. Recent studies show that primary liver tumors display heterogeneity similar to that observed in the normal tissue providing clues to the cellular precursor of the tumor and how variations in the lobule microenvironment support tumor formation and aggressiveness. Identifying the principles that control cellular diversity in a healthy liver may highlight potential mechanisms driving hepatic tumor heterogeneity.

LKB1 acts as a critical brake for the glucagon-mediated fasting response.

As important as the fasting response is for survival, an inability to shut it down once nutrients become available can lead to exacerbated disease and severe wasting. The liver is central to transitions between feeding and fasting states, with glucagon being a key initiator of the hepatic fasting response. However, the precise mechanisms controlling fasting are not well defined. One potential mediator of these transitions is liver kinase B1 (LKB1), given its role in nutrient sensing. Here, we show LKB1 knockout mice have a severe wasting and prolonged fasting phenotype despite increased food intake. By applying RNA sequencing and intravital microscopy, we show that loss of LKB1 leads to a dramatic reprogramming of the hepatic lobule through robust up-regulation of periportal genes and functions. This is likely mediated through the opposing effect that LKB1 has on glucagon pathways and gene expression. Conclusion: Our findings show that LKB1 acts as a brake to the glucagon-mediated fasting response, resulting in "periportalization" of the hepatic lobule and whole-body metabolic inefficiency. These findings reveal a mechanism by which hepatic metabolic compartmentalization is regulated by nutrient-sensing.

Hepatocyte-specific eNOS deletion impairs exercise-induced adaptations in hepatic mitochondrial function and autophagy.

OBJECTIVE: Endothelial nitric oxide synthase (eNOS) is a potential mediator of exercise-induced hepatic mitochondrial adaptations. METHODS: Here, male and female hepatocyte-specific eNOS knockout (eNOShep-/- ) and intact hepatic eNOS (eNOSfl/fl ) mice performed voluntary wheel-running exercise (EX) or remained in sedentary cage conditions for 10 weeks. RESULTS: EX resolved the exacerbated hepatic steatosis in eNOShep-/- male mice. Elevated hydrogen peroxide emission (~50% higher in eNOShep-/- vs. eNOSfl/fl mice) was completely ablated with EX. Interestingly, EX increased [1-14 C] palmitate oxidation in eNOSfl/fl male mice, but this was blunted in the eNOShep-/- male mice. eNOShep-/- mice had lower markers of the energy sensors AMP-activated protein kinase (AMPK)/phospho- (p)AMPK and mammalian target of rapamycin (mTOR) and p-mTOR, as well as the autophagy initiators serine/threonine-protein kinase ULK1 and pULK1, compared with eNOSfl/fl mice. Females showed elevated electron transport chain protein content and markers of mitochondrial biogenesis (transcription factor A, mitochondrial, peroxisome proliferator-activated receptor-gamma coactivator 1α). CONCLUSIONS: Collectively, this study demonstrates for the first time, to the authors' knowledge, the requirement of eNOS in hepatocytes in the EX-induced increases in hepatic fatty acid oxidation in male mice. Deletion of eNOS in hepatocytes also appears to impair the energy-sensing ability of the cell and inhibit the activation of the autophagy initiating factor ULK1. These data uncover the important and novel role of hepatocyte eNOS in EX-induced hepatic mitochondrial adaptations.

Compromised hepatic mitochondrial fatty acid oxidation and reduced markers of mitochondrial turnover in human NAFLD.

BACKGROUND AND AIMS: NAFLD and its more-advanced form, steatohepatitis (NASH), is associated with obesity and is an independent risk factor for cardiovascular, liver-related, and all-cause mortality. Available human data examining hepatic mitochondrial fatty acid oxidation (FAO) and hepatic mitochondrial turnover in NAFLD and NASH are scant. APPROACH AND RESULTS: To investigate this relationship, liver biopsies were obtained from patients with obesity undergoing bariatric surgery and data clustered into four groups based on hepatic histopathological classification: Control (CTRL; no disease); NAFL (steatosis only); Borderline-NASH (steatosis with lobular inflammation or hepatocellular ballooning); and Definite-NASH (D-NASH; steatosis, lobular inflammation, and hepatocellular ballooning). Hepatic mitochondrial complete FAO to CO2 and the rate-limiting enzyme in ß-oxidation (ß-hydroxyacyl-CoA dehydrogenase activity) were reduced by ~40%-50% with D-NASH compared with CTRL. This corresponded with increased hepatic mitochondrial reactive oxygen species production, as well as dramatic reductions in markers of mitochondrial biogenesis, autophagy, mitophagy, fission, and fusion in NAFL and NASH. CONCLUSIONS: These findings suggest that compromised hepatic FAO and mitochondrial turnover are intimately linked to increasing NAFLD severity in patients with obesity.

Liver Zonation - Revisiting Old Questions With New Technologies.

Despite the ever-increasing prevalence of non-alcoholic fatty liver disease (NAFLD), the etiology and pathogenesis remain poorly understood. This is due, in part, to the liver's complex physiology and architecture. The liver maintains glucose and lipid homeostasis by coordinating numerous metabolic processes with great efficiency. This is made possible by the spatial compartmentalization of metabolic pathways a phenomenon known as liver zonation. Despite the importance of zonation to normal liver function, it is unresolved if and how perturbations to liver zonation can drive hepatic pathophysiology and NAFLD development. While hepatocyte heterogeneity has been identified over a century ago, its examination had been severely hindered due to technological limitations. Recent advances in single cell analysis and imaging technologies now permit further characterization of cells across the liver lobule. This review summarizes the advances in examining liver zonation and elucidating its regulatory role in liver physiology and pathology. Understanding the spatial organization of metabolism is vital to further our knowledge of liver disease and to provide targeted therapeutic avenues.

Critical Role for Hepatocyte-Specific eNOS in NAFLD and NASH.

Regulation of endothelial nitric oxide synthase (eNOS) in hepatocytes may be an important target in nonalcoholic fatty liver disease (NAFLD) development and progression to nonalcoholic steatohepatitis (NASH). In this study, we show genetic deletion and viral knockdown of hepatocyte-specific eNOS exacerbated hepatic steatosis and inflammation, decreased hepatic mitochondrial fatty acid oxidation and respiration, increased mitochondrial H2O2 emission, and impaired the hepatic mitophagic (BNIP3 and LC3II) response. Conversely, overexpressing eNOS in hepatocytes in vitro and in vivo increased hepatocyte mitochondrial respiration and attenuated Western diet-induced NASH. Moreover, patients with elevated NAFLD activity score (histology score of worsening steatosis, hepatocyte ballooning, and inflammation) exhibited reduced hepatic eNOS expression, which correlated with reduced hepatic mitochondrial fatty acid oxidation and lower hepatic protein expression of mitophagy protein BNIP3. The current study reveals an important molecular role for hepatocyte-specific eNOS as a key regulator of NAFLD/NASH susceptibility and mitochondrial quality control with direct clinical correlation to patients with NASH.

A dietary ketone ester mitigates histological outcomes of NAFLD and markers of fibrosis in high-fat diet fed mice.

Nutritional ketosis as a therapeutic tool has been extended to the treatment of metabolic diseases, including obesity, type 2 diabetes, and nonalcoholic fatty liver disease (NAFLD). The purpose of this study was to determine whether dietary administration of the ketone ester (KE) R,S-1,3-butanediol diacetoacetate (BD-AcAc2) attenuates markers of hepatic stellate cell (HSC) activation and hepatic fibrosis in the context of high-fat diet (HFD)-induced obesity. Six-week-old male C57BL/6J mice were placed on a 10-wk ad libitum HFD (45% fat, 32% carbohydrates, 23% proteins). Mice were then randomized to one of three groups (n = 10 per group) for an additional 12 wk: 1) control (CON), continuous HFD; 2) pair-fed (PF) to KE, and 3) KE (HFD + 30% energy from BD-AcAc2, KE). KE feeding significantly reduced histological steatosis, inflammation, and total NAFLD activity score versus CON, beyond improvements observed for calorie restriction alone (PF). Dietary KE supplementation also reduced the protein content and gene expression of profibrotic markers (α-SMA, COL1A1, PDGF-ß, MMP9) versus CON (P < 0.05), beyond reductions observed for PF versus CON. Furthermore, KE feeding increased hepatic markers of anti-inflammatory M2 macrophages (CD163) and also reduced proinflammatory markers [tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and cellular communication network factor 1 (CCN1)] versus CON and PF (P ≤ 0.05), in the absence of changes in markers of total hepatic macrophage content (F4/80 and CD68; P > 0.05). These data highlight that the dietary ketone ester BD-AcAc2 ameliorates histological NAFLD and inflammation and reduces profibrotic and proinflammatory markers. Future studies to further explore potential mechanisms are warranted.NEW & NOTEWORTHY To our knowledge, this is the first study focusing on hepatic outcomes in response to dietary ketone ester feeding in male mice with HFD-induced NAFLD. Novel findings include that dietary ketone ester feeding ameliorates NAFLD outcomes via reductions in histological steatosis and inflammation. These improvements were beyond those observed for caloric restriction alone. Furthermore, dietary ketone ester feeding was associated with greater reductions in markers of hepatic fibrogenesis and inflammation compared with control and calorie-restricted mice.

A Fad too Far? Dietary Strategies for the Prevention and Treatment of NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is a major health problem, and its prevalence has increased in recent years, concurrent with rising rates of obesity and other metabolic diseases. Currently, there are no FDA-approved pharmacological therapies for NAFLD, and lifestyle interventions, including weight loss and exercise, remain the cornerstones for treatment. Manipulating diet composition and eating patterns may be a sustainable approach to NAFLD treatment. Dietary strategies including Paleolithic, ketogenic, Mediterranean, high-protein, plant-based, low-carbohydrate, and intermittent fasting diets have become increasingly popular because of their purported benefits on metabolic disease. This review highlights what is currently known about these popular dietary approaches in the management of NAFLD in clinical populations with mechanistic insight from animal studies. It also identifies key knowledge gaps to better inform future preclinical and clinical studies aimed at the treatment of NAFLD.

The Emerging Role of Hepatocellular eNOS in Non-alcoholic Fatty Liver Disease Development.

Non-alcoholic fatty liver disease (NAFLD) is comprised of a spectrum of liver injury ranging from excess fat accumulation in the liver (steatosis), to steatohepatitis (NASH), to its end stage of cirrhosis. A hallmark of NAFLD progression is the decline in function of hepatic mitochondria, although the mechanisms remain unresolved. Given the important role endothelial nitric oxide synthase (eNOS) plays in mitochondrial dynamics in other tissues, it has emerged as a potential mediator of maintaining mitochondrial function in the liver. In this mini review, we summarize the most relevant findings that extends current understanding of eNOS as a regulator of mitochondrial biogenesis, and identifies a potential additional role in mitochondrial turnover and attenuating inflammation during NAFLD development and progression.

Ketogenic diet in combination with voluntary exercise impacts markers of hepatic metabolism and oxidative stress in male and female Wistar rats.

Ketogenic diets (KDs) are shown to benefit hepatic metabolism; however, their effect on the liver when combined with exercise is unknown. We investigated the effects of a KD versus a "western" diet (WD) on markers of hepatic lipid metabolism and oxidative stress in exercising rats. Male and female Wistar rats with access to voluntary running wheels were randomized to 3 groups (n = 8-14 per group): standard chow (SC; 17% fat), WD (42% fat), or KD (90.5% fat) for 7 weeks. Body fat percentage (BF%) was increased in WD and KD versus SC, although KD females displayed lower BF% versus WD (p ≤ 0.05). Liver triglycerides were higher in KD and WD versus SC but were attenuated in KD females versus WD (p ≤ 0.05). KD suppressed hepatic markers of de novo lipogenesis (fatty acid synthase, acetyl coenzyme A carboxylase) and increased markers of mitochondrial biogenesis/content (peroxisome proliferator activated receptor-1α, mitochondrial transcription factor A (TFAM), and citrate synthase activity). KD also increased hepatic glutathione peroxidase 1 and lowered oxidized glutathione. Female rats exhibited elevated hepatic markers of mitochondrial biogenesis (TFAM), mitophagy (light chain 3 II/I ratio, autophagy-related protein 12:5), and cellular energy homeostasis (phosphorylated 5'AMP-activated protein kinase/5'AMP-activated protein kinase) versus males. These data highlight that KD and exercise beneficially impacts hepatic metabolism and oxidative stress and merits further investigation. Novelty KD feeding combined with exercise improved hepatic oxidative stress, suppressed markers of de novo lipogenesis, and increased markers of mitochondrial content versus WD feeding. Males and females responded similarly to combined KD feeding and exercise. Female rats exhibited elevated hepatic markers of autophagy/mitophagy and energy homeostasis compared with male rats.

eNOS deletion impairs mitochondrial quality control and exacerbates Western diet-induced NASH.

Dysregulated mitochondrial quality control leads to mitochondrial functional impairments that are central to the development and progression of hepatic steatosis to nonalcoholic steatohepatitis (NASH). Here, we identify hepatocellular localized endothelial nitric oxide synthase (eNOS) as a novel master regulator of mitochondrial quality control. Mice lacking eNOS were more susceptible to Western diet-induced hepatic inflammation and fibrosis in conjunction with decreased markers of mitochondrial biogenesis and turnover. The hepatocyte-specific influence was verified via magnetic activated cell sorting purified primary hepatocytes and in vitro siRNA-induced knockdown of eNOS. Hepatic mitochondria from eNOS knockout mice revealed decreased markers of mitochondrial biogenesis (PPARγ coactivator-1α, mitochondrial transcription factor A) and autophagy/mitophagy [BCL-2-interacting protein-3 (BNIP3), 1A/1B light chain 3B (LC3)], suggesting decreased mitochondrial turnover rate. eNOS knockout in primary hepatocytes exhibited reduced fatty acid oxidation capacity and were unable to mount a normal BNIP3 response to a mitophagic challenge compared with wild-type mice. Finally, we demonstrate that eNOS is required in primary hepatocytes to induce activation of the stress-responsive transcription factor nuclear factor erythroid 2-related factor 2 (NRF2). Thus, our data demonstrate that eNOS is an important regulator of hepatic mitochondrial content and function and NASH susceptibility.

Compromised Exercise Capacity and Mitochondrial Dysfunction in the Osteogenesis Imperfecta Murine (oim) Mouse Model.

Osteogenesis imperfecta (OI) is a heritable connective tissue disorder that most often arises from type I collagen-COL1A1 and COL1A2-gene defects leading to skeletal fragility, short stature, blue-gray sclera, and muscle weakness. Relative to the skeletal fragility, muscle weakness is much less understood. Recent investigations into OI muscle weakness in both patients and mouse models have revealed the presence of an inherent muscle pathology. Understanding the mechanisms responsible for OI muscle weakness is critical, particularly in light of the extensive cross-talk between muscle and bone via mechanotransduction and biochemical signaling. In the following study we initially subjected WT and oim/oim mice, modeling severe human OI type III, to either weight-bearing (voluntary wheel-running) or non-weight-bearing (swimming) exercise regimens as a modality to improve muscle strength and ultimately bone strength. The oim/oim mice ran only 35% to 42% of the distance run by age- and sex-matched WT mice and exhibited little improvement with either exercise regimen. Upon further investigation, we determined that oim/oim gastrocnemius muscle exhibited severe mitochondrial dysfunction as characterized by a 52% to 65% decrease in mitochondrial respiration rates, alterations in markers of mitochondrial biogenesis, mitophagy, and the electron transport chain components, as well as decreased mitochondrial citrate synthase activity, relative to age- and sex-matched WT gastrocnemius muscle. Thus, mitochondrial dysfunction in the oim/oim mouse likely contributes to compromised muscle function and reduced physical activity levels. © 2019 American Society for Bone and Mineral Research.

Estrogen receptor-α signaling maintains immunometabolic function in males and is obligatory for exercise-induced amelioration of nonalcoholic fatty liver.

The role of estrogen receptor-α (ERα) signaling in immunometabolic function is established in females. However, its necessity in males, while appreciated, requires further study. Accordingly, we first determined whether lower metabolic function in male mice compared with females is related to reduced ERα expression. ERα protein expression in metabolically active tissues was lower in males than in females, and this lower expression was associated with worse glucose tolerance. Second, we determined whether ERα is required for optimal immunometabolic function in male mice consuming a chow diet. Despite lower expression of ERα in males, its genetic ablation (KO) caused an insulin-resistant phenotype characterized by enhanced adiposity, glucose intolerance, hepatic steatosis, and metaflammation in adipose tissue and liver. Last, we determined whether ERα is essential for exercise-induced metabolic adaptations. Twelve-week-old wild-type (WT) and ERα KO mice either remained sedentary (SED) or were given access to running wheels (WR) for 10 wk while fed an obesogenic diet. Body weight and fat mass were lower in WR mice regardless of genotype. Daily exercise obliterated immune cell infiltration and inflammatory gene transcripts in adipose tissue in both genotypes. In the liver, however, wheel running suppressed hepatic steatosis and inflammatory gene transcripts in WT but not in KO mice. In conclusion, the present findings indicate that ERα is required for optimal immunometabolic function in male mice despite their reduced ERα protein expression in metabolically active tissues. Furthermore, for the first time, we show that ERα signaling appears to be obligatory for exercise-induced prevention of hepatic steatosis.

Curcumin supplementation mitigates NASH development and progression in female Wistar rats.

Curcumin, a naturally occurring plant polyphenolic compound, may have beneficial effects in nonalcoholic steatohepatitis (NASH) development. We examined whether curcumin supplementation could be used in both prevention and treatment of NASH with fibrosis. Female Wistar rats were provided ad libitum access to a "western diet" (WD) high in fat (43% kcal), sucrose (29% kcal), and cholesterol (2% w/v), as well as 15% fructose drinking water. Intraperitoneal CC14 injections (0.5 mL/kg) were also administered at weeks 1, 2, 4, and 6 to accelerate development of a NASH with fibrosis phenotype. Rats were randomized to four groups (n = 9-12/group) and fed ad libitum: (1) WD for 8-weeks (8WD), (2) WD enriched with curcumin for 8-weeks (8WD+C; 0.2% curcumin, BCM-95, DolCas Biotech) to assess prevention, (3) WD for 12-weeks (12WD), (4) WD for 8-weeks followed by 4-weeks WD+C (12WD+C) to assess treatment. Curcumin prevention (8WD vs. 8WD+C) attenuated (P < 0.05) histological liver inflammation, molecular markers of fibrosis (Col1a1 mRNA) and a serum marker of liver injury (AST). Curcumin treatment (12WD vs. 12WD+C) reduced (P < 0.05) hepatocellular inflammation, steatosis, NAFLD Activity Scores, and serum markers of liver injury (AST, ALP). Moreover, curcumin treatment also increased hepatic pACC/ACC, ApoB100, and SOD1 protein, and decreased hepatic FGF-21 levels; whereas, curcumin prevention increased hepatic glutathione levels. Both curcumin prevention and treatment reduced molecular markers of hepatic fibrosis (Col1a1 mRNA) and inflammation (TNF-α, SPP1 mRNA). Curcumin supplementation beneficially altered the NASH phenotype in female Wistar rats, particularly the reversal of hepatocellular inflammation.

Maternal Physical Activity and Sex Impact Markers of Hepatic Mitochondrial Health.

INTRODUCTION: Maternal exercise and physical activity during the gestational period can be protective against maternal high-fat diet-induced hepatic steatosis in older offspring. However, it is unknown whether these protective effects are seen in younger offspring. In this study, we investigated whether maternal physical activity would attenuate maternal western diet (WD)-induced steatosis in young adult rats. METHODS: Female Wistar rats (7-8 wk of age) were randomized into WD (42% fat, 27% sucrose) or normal chow diet (ND), and further randomized into physical activity (RUN) or sedentary (SED) conditions for a total of four groups. Dams returned to ND/SED conditions after parturition. Postweaning, offspring were maintained in ND/SED conditions for 18 wk. RESULTS: Maternal WD-induced increases in male offspring body mass was attenuated in the WD/RUN offspring (P < 0.05). Maternal WD feeding significantly increased hepatic steatosis in male (but not female offspring), which was not attenuated by maternal RUN. However, maternal RUN increased (P < 0.05) hepatic markers of mitochondrial biogenesis and mitophagy (mitochondrial transcription factor A, peroxisome proliferator activator receptor γ, and nuclear factor E2-related factor 2) in all offspring and the mitophagy marker BCL2-interacting protein 3 in WD/RUN offspring. Interestingly, hepatic markers of de novo lipogenesis (fatty acid synthase and acetyl coenzyme A carboxylase), mitophagy (autophagy-related gene 12:5, BCL2-interacting protein 3, P62, and LC3 II/I), and mitochondria biogenesis/content (mitochondrial transcription factor A and OXPHOS-Complex II) were significantly increased in female versus male offspring. CONCLUSION: Although maternal physical activity did not attenuate maternal WD-induced hepatic steatosis as has been previously reported in older adult offspring, it did significantly increase hepatic markers of mitochondrial biogenesis and mitophagy. Furthermore, female offspring had elevated hepatic markers of mitochondrial health, possibly explaining why female rats are protected against maternal WD-induced hepatic steatosis. Future studies are warranted to shed light on the time line of hepatic steatosis development under the influence of maternal physical activity.