Vitamin B12 and folate decrease inflammation and fibrosis in NASH by preventing syntaxin 17 homocysteinylation.

BACKGROUND & AIMS: Several recent clinical studies have shown that serum homocysteine (Hcy) levels are positively correlated, while vitamin B12 (B12) and folate levels are negative correlated, with non-alcoholic steatohepatitis (NASH) severity. However, it is not known whether hyperhomocysteinemia (HHcy) plays a pathogenic role in NASH. METHODS: We examined the effects of HHcy on NASH progression, metabolism, and autophagy in dietary and genetic mouse models, patients, and primates. We employed vitamin B12 (B12) and folate (Fol) to reverse NASH features in mice and cell culture. RESULTS: Serum Hcy correlated with hepatic inflammation and fibrosis in NASH. Elevated hepatic Hcy induced and exacerbated NASH. Gene expression of hepatic Hcy-metabolizing enzymes was downregulated in NASH. Surprisingly, we found increased homocysteinylation (Hcy-lation) and ubiquitination of multiple hepatic proteins in NASH including the key autophagosome/lysosome fusion protein, Syntaxin 17 (Stx17). This protein was Hcy-lated and ubiquitinated, and its degradation led to a block in autophagy. Genetic manipulation of Stx17 revealed its critical role in regulating autophagy, inflammation and fibrosis during HHcy. Remarkably, dietary B12/Fol, which promotes enzymatic conversion of Hcy to methionine, decreased HHcy and hepatic Hcy-lated protein levels, restored Stx17 expression and autophagy, stimulated ß -oxidation of fatty acids, and improved hepatic histology in mice with pre-established NASH. CONCLUSIONS: HHcy plays a key role in the pathogenesis of NASH via Stx17 homocysteinylation. B12/folate also may represent a novel first-line therapy for NASH. LAY SUMMARY: The incidence of non-alcoholic steatohepatitis, for which there are no approved pharmacological therapies, is increasing, posing a significant healthcare challenge. Herein, based on studies in mice, primates and humans, we found that dietary supplementation with vitamin B12 and folate could have therapeutic potential for the prevention or treatment of non-alcoholic steatohepatitis.

Negative regulation of AMPK signaling by high glucose via E3 ubiquitin ligase MG53.

As a master regulator of metabolism, AMP-activated protein kinase (AMPK) is activated upon energy and glucose shortage but suppressed upon overnutrition. Exaggerated negative regulation of AMPK signaling by nutrient overload plays a crucial role in metabolic diseases. However, the mechanism underlying the negative regulation is poorly understood. Here, we demonstrate that high glucose represses AMPK signaling via MG53 (also called TRIM72) E3-ubiquitin-ligase-mediated AMPKα degradation and deactivation. Specifically, high-glucose-stimulated reactive oxygen species (ROS) signals AKT to phosphorylate AMPKα at S485/491, which facilitates the recruitment of MG53 and the subsequent ubiquitination and degradation of AMPKα. In addition, high glucose deactivates AMPK by ROS-dependent suppression of phosphorylation of AMPKα at T172. These findings not only delineate the mechanism underlying the impairment of AMPK signaling in overnutrition-related diseases but also highlight the significance of keeping the yin-yang balance of AMPK signaling in the maintenance of metabolic homeostasis.

Chemoproteomics reveals baicalin activates hepatic CPT1 to ameliorate diet-induced obesity and hepatic steatosis.

Obesity and related metabolic diseases are becoming worldwide epidemics that lead to increased death rates and heavy health care costs. Effective treatment options have not been found yet. Here, based on the observation that baicalin, a flavonoid from the herbal medicine Scutellaria baicalensis, has unique antisteatosis activity, we performed quantitative chemoproteomic profiling and identified carnitine palmitoyltransferase 1 (CPT1), the controlling enzyme for fatty acid oxidation, as the key target of baicalin. The flavonoid directly activated hepatic CPT1 with isoform selectivity to accelerate the lipid influx into mitochondria for oxidation. Chronic treatment of baicalin ameliorated diet-induced obesity (DIO) and hepatic steatosis and led to systemic improvement of other metabolic disorders. Disruption of the predicted binding site of baicalin on CPT1 completely abolished the beneficial effect of the flavonoid. Our discovery of baicalin as an allosteric CPT1 activator opens new opportunities for pharmacological treatment of DIO and associated sequelae.

Eplerenone restores 24-h blood pressure circadian rhythm and reduces advanced glycation end-products in rhesus macaques with spontaneous hypertensive metabolic syndrome.

Hypertension is often associated with metabolic syndrome (MetS), and serves as a risk factor of MetS and its complications. Blood pressure circadian rhythm in hypertensive patients has been suggested to contribute to cardiovascular consequences and organ damage of hypertension. But circadian changes of BP and their response to drugs have not been clearly investigated in non-human primates (NHPs) of MetS with hypertension. Here, we identified 16 elderly, hypertensive MetS rhesus monkeys from our in-house cohort. With implanted telemetry, we investigate BP changes and its circadian rhythm, together with the effect of antihypertensive drugs on BP and its diurnal fluctuation. MetS hypertensive monkeys displayed higher BP, obesity, glucose intolerance, and dyslipidemia. We also confirmed impaired 24-h BP circadian rhythm in MetS hypertensive monkeys. Importantly, Eplerenone, a mineralocorticoid receptor blocker, exerts multiple beneficial effects in MetS hypertensive monkeys, including BP reduction, 24-h BP circadian rhythm restoration, and decreased plasma concentration of inflammation factors and advanced glycation end-products. In summary, we identified a naturally-developed hypertensive MetS NHP model, which is of great value in the studies on pathogenesis of MetS-associated hypertension and development of novel therapeutic strategies. We also provided multiple novel mechanistic insights of the beneficial effect of Eplerenone on MetS with hypertension.

The N-glycoform of sRAGE is the key determinant for its therapeutic efficacy to attenuate injury-elicited arterial inflammation and neointimal growth.

UNLABELLED: Signaling of the receptor for advanced glycation end products (RAGE) has been implicated in the development of injury-elicited vascular complications. Soluble RAGE (sRAGE) acts as a decoy of RAGE and has been used to treat pathological vascular conditions in animal models. However, previous studies used a high dose of sRAGE produced in insect Sf9 cells (sRAGE(Sf9))and multiple injections to achieve the therapeutic outcome. Here, we explore whether modulation of sRAGE N-glycoform impacts its bioactivity and augments its therapeutic efficacy. We first profiled carbohydrate components of sRAGE produced in Chinese hamster Ovary cells (sRAGE(CHO)) to show that a majority of its N-glycans belong to sialylated complex types that are not shared by sRAGE(Sf9). In cell-based NF-κB activation and vascular smooth muscle cell (VSMC) migration assays, sRAGE(CHO) exhibited a significantly higher bioactivity relative to sRAGE(Sf9) to inhibit RAGE alarmin ligand-induced NF-κB activation and VSMC migration. We next studied whether this N-glycoform-associated bioactivity of sRAGE(CHO) is translated to higher in vivo therapeutic efficacy in a rat carotid artery balloon injury model. Consistent with the observed higher bioactivity in cell assays, sRAGE(CHO) significantly reduced injury-induced neointimal growth and the expression of inflammatory markers in injured vasculature. Specifically, a single dose of 3 ng/g of sRAGE(CHO) reduced neointimal hyperplasia by over 70%, whereas the same dose of sRAGE(Sf9) showed no effect. The administered sRAGE(CHO) is rapidly and specifically recruited to the injured arterial locus, suggesting that early intervention of arterial injury with sRAGE(CHO) may offset an inflammatory circuit and reduce the ensuing tissue remodeling. Our findings showed that the N-glycoform of sRAGE is the key determinant underlying its bioactivity and thus is an important glycobioengineering target to develop a highly potent therapeutic sRAGE for future clinical applications. KEY MESSAGE: The specific N-glycoform modification is the key underlying sRAGE bioactivity Markedly reduced sRAGE dose to attenuate neointimal hyperplasia and inflammation Provide a molecular target for glycobioengineering of sRAGE as a therapeutic protein Blocking RAGE alarmin ligands during acute injury phase offsets neointimal growth.