Conditional deletion of CEACAM1 causes hepatic stellate cell activation.

Objectives: Hepatic CEACAM1 expression declines with advanced hepatic fibrosis stage in patients with MASH. Global and hepatocyte-specific deletions of Ceacam1 impair insulin clearance to cause hepatic insulin resistance and steatosis. They also cause hepatic inflammation and fibrosis, a condition characterized by excessive collagen production from activated hepatic stellate cells (HSCs). Given the positive effect of PPARγ on CEACAM1 transcriptoin and on HSCs quiescence, the current studies investigated whether CEACAM1 loss from HSCs causes their activation. Methods: We examined whether lentiviral shRNA-mediated CEACAM1 donwregulation (KD-LX2) activates cultured human LX2 stellate cells. We also generated LratCre+Cc1 fl/fl mutants with conditional Ceacam1 deletion in HSCs and characterized their MASH phenotype. Media transfer experiments were employed to examine whether media from mutant human and murine HSCs activate their wild-type counterparts. Results: LratCre+Cc1 fl/fl mutants displayed hepatic inflammation and fibrosis but without insulin resistance or hepatic steatosis. Their HSCs, like KD-LX2 cells, underwent myofibroblastic transformation and their media activated wild-type HDCs. This was inhibited by nicotinic acid treatment which stemmed the release of IL-6 and fatty acids, both of which activate the epidermal growth factor receptor (EGFR) tyrosine kinase. Gefitinib inhibition of EGFR and its downstream NF-κB/IL-6/STAT3 inflammatory and MAPK-proliferation pathways also blunted HSCs activation in the absence of CEACAM1. Conclusions: Loss of CEACAM1 in HSCs provoked their myofibroblastic transformation in the absence of insulin resistance and hepatic steatosis. This response is mediated by autocrine HSCs activation of the EGFR pathway that amplifies inflammation and proliferation.

The Gut Microbiome Responds Progressively to Fat and/or Sugar-Rich Diets and Is Differentially Modified by Dietary Fat and Sugar.

Describing diet-related effects on the gut microbiome is essential for understanding its interactions with fat and/or sugar-rich diets to promote obesity-related metabolic diseases. Here, we sequenced the V3-V4 hypervariable region of the bacterial 16S rRNA gene to study the composition and dynamics of the gut microbiome of adult mice fed diets rich in fat and/or sugar, at 9 and 18 weeks of diet. Under high-fat, high-sugar diet, the abundances of Tuzzerella and Anaerovorax were transiently increased at 9 weeks, while Lactobacillus remained elevated at 9 and 18 weeks. The same diet decreased the abundances of Akkermansia, Paludicola, Eisenbergiella, and Butyricicoccus at 9 and 18 weeks, while Intestinimonas and UCG-009 of the Butyricicoccaceae family responded only at 18 weeks. The high-fat diet decreased the abundances of UBA1819 at 9 weeks, and Gastranaerophilales, Clostridia UCG-014, and ASF356 at 9 and 18 weeks. Those of Marvinbryantia, Harryflintia, Alistipes, Blautia, Lachnospiraceae A2, Eubacterium coprostanoligenes group, and Eubacterium brachy group were lowered only at 18 weeks. Interestingly, these genera were not sensitive to the high-sugar diet. The mouse gut microbiome was differentially affected by diets rich in fat or fat and sugar. The differences observed at 9 and 18 weeks indicate a progressive microbiome response.

Enrichment of hepatic glycogen and plasma glucose from H218 O informs gluconeogenic and indirect pathway fluxes in naturally feeding mice.

Deuterated water (2 H2 O) is a widely used tracer of carbohydrate biosynthesis in both preclinical and clinical settings, but the significant kinetic isotope effects (KIE) of 2 H can distort metabolic information and mediate toxicity. 18 O-water (H2 18 O) has no significant KIE and is incorporated into specific carbohydrate oxygens via well-defined mechanisms, but to date it has not been evaluated in any animal model. Mice were given H2 18 O during overnight feeding and 18 O-enrichments of liver glycogen, triglyceride glycerol (TG), and blood glucose were quantified by 13 C NMR and mass spectrometry (MS). Enrichment of oxygens 5 and 6 relative to body water informed indirect pathway contributions from the Krebs cycle and triose phosphate sources. Compared with mice fed normal chow (NC), mice whose NC was supplemented with a fructose/glucose mix (i.e., a high sugar [HS] diet) had significantly higher indirect pathway contributions from triose phosphate sources, consistent with fructose glycogenesis. Blood glucose and liver TG 18 O-enrichments were quantified by MS. Blood glucose 18 O-enrichment was significantly higher for HS versus NC mice and was consistent with gluconeogenic fructose metabolism. TG 18 O-enrichment was extensive for both NC and HS mice, indicating a high turnover of liver triglyceride, independent of diet. Thus H2 18 O informs hepatic carbohydrate biosynthesis in similar detail to 2 H2 O but without KIE-associated risks.

Integration of Liver Glycogen and Triglyceride NMR Isotopomer Analyses Provides a Comprehensive Coverage of Hepatic Glucose and Fructose Metabolism.

Dietary glucose and fructose are both efficiently assimilated by the liver but a comprehensive measurement of this process starting from their conversion to sugar phosphates, involvement of the pentose phosphate pathway (PPP), and conversion to glycogen and lipid storage products, remains incomplete. Mice were fed a chow diet supplemented with 35 g/100 mL drinking water of a 55/45 fructose/glucose mixture for 18 weeks. On the final night, the sugar mixture was enriched with either [U-13C]glucose or [U-13C]fructose, and deuterated water (2H2O) was also administered. 13C-isotopomers representing newly synthesized hepatic glucose-6-phosphate (glucose-6-P), glycerol-3-phosphate, and lipogenic acetyl-CoA were quantified by 2H and 13C NMR analysis of post-mortem liver glycogen and triglyceride. These data were applied to a metabolic model covering glucose-6-P, PPP, triose-P, and de novo lipogenesis (DNL) fluxes. The glucose supplement was converted to glucose-6-P via the direct pathway, while the fructose supplement was metabolized by the liver to gluconeogenic triose-P via fructokinase-aldolase-triokinase. Glucose-6-P from all carbohydrate sources accounted for 40-60% of lipogenic acetyl-CoA and 10-12% was oxidized by the pentose phosphate pathway (PPP). The yield of NADPH from PPP flux accounted for a minority (~30%) of the total DNL requirement. In conclusion, this approach integrates measurements of glucose-6-P, PPP, and DNL fluxes to provide a holistic and informative assessment of hepatic glucose and fructose metabolism.

Determining the contribution of a high-fructose corn syrup formulation to hepatic glycogen synthesis during ad-libitum feeding in mice.

Excessive sugar intake including high-fructose corn syrup (HFCS) is implicated in the rise of obesity, insulin resistance and non-alcoholic fatty liver disease. Liver glycogen synthesis is influenced by both fructose and insulin signaling. Therefore, the effect of HFCS on hepatic glycogenesis was evaluated in mice feeding ad-libitum. Using deuterated water: the fraction of glycogen derived from triose-P sources, Krebs cycle substrates, and direct pathway + cycling, was measured in 9 normal-chow fed mice (NC) and 12 mice fed normal chow plus a 55% fructose/45% glucose mix in the drinking water at 30% w/v (HFCS-55). This was enriched with [U-13C]fructose or [U-13C]glucose to determine the contribution of each to glycogenesis. For NC, direct pathway + cycling, Krebs cycle, and triose-P sources accounted for 66 ± 0.7%, 23 ± 0.8% and 11 ± 0.4% of glycogen synthesis, respectively. HFCS-55 mice had similar direct pathway + cycling (64 ± 1%) but lower Krebs cycle (12 ± 1%, p < 0.001) and higher triose-P contributions (24 ± 1%, p < 0.001). HFCS-55-fructose contributed 17 ± 1% via triose-P and 2 ± 0% via Krebs cycle. HFCS-55-glucose contributed 16 ± 3% via direct pathway and 1 ± 0% via Krebs cycle. In conclusion, HFCS-55 supplementation resulted in similar hepatic glycogen deposition rates. Indirect pathway contributions shifted from Krebs cycle to Triose-P sources reflecting HFCS-55-fructose utilization, while HFCS-55-glucose was incorporated almost exclusively by the direct pathway.

Estimating pentose phosphate pathway activity from the analysis of hepatic glycogen 13 C-isotopomers derived from [U-13 C]fructose and [U-13 C]glucose.

PURPOSE: The pentose phosphate pathway (PPP) is an important component of hepatic intermediary metabolism. Jin et al developed an elegant 13 C-NMR method for measuring hepatic PPP flux by quantifying the distribution of glucose 13 C-isotopomers formed from [U-13 C]glycerol. We demonstrate that this approach can be extended to exogenous [U-13 C]fructose and [U-13 C]glucose precursors by 13 C-NMR analysis of glycogen. METHODS: Twelve male C57BL/6 mice fed standard chow were provided a 55/45 mixture of fructose and glucose at 30% w/v in the drinking water for 18 wk. On the evening before sacrifice, the fructose component was enriched with 20% [U-13 C]fructose for 6 mice, while the glucose component was enriched with 20% [U-13 C]glucose for the remaining 6 mice. Mice were allowed to feed and drink naturally overnight, and then, euthanized. Livers were freeze-clamped and glycogen was extracted and derivatized for 13 C NMR spectroscopy. Flux of each sugar into the PPP relative to its incorporation into glycogen was quantified from selected 13 C glycogen isotopomer ratios. RESULTS: Both [U-13 C]fructose and [U-13 C]glucose precursors yielded glycogen 13 C-isotopomer distributions that were characteristic of PPP activity. The fraction of [U-13 C]glucose utilized by the PPP relative to its conversion to glycogen via the direct pathway was 14 ± 1%, while that from [U-13 C]fructose relative to its conversion to glycogen via the indirect pathway was significantly lower (10 ± 1%, P = .00032). CONCLUSIONS: Hepatic PPP fluxes from both [U-13 C]glucose and [U-13 C]fructose precursors were assessed by 13 C NMR analysis of glycogen 13 C-isotopomers. Glucose-6-phosphate generated via glucokinase and the direct pathway is preferentially utilized by the PPP.

A Mitochondrial Approach to Cardiovascular Risk and Disease.

BACKGROUND: Cardiovascular diseases (CVDs) are a leading risk factor for mortality worldwide and the number of CVDs victims is predicted to rise through 2030. While several external parameters (genetic, behavioral, environmental and physiological) contribute to cardiovascular morbidity and mortality; intrinsic metabolic and functional determinants such as insulin resistance, hyperglycemia, inflammation, high blood pressure and dyslipidemia are considered to be dominant factors. METHODS: Pubmed searches were performed using different keywords related with mitochondria and cardiovascular disease and risk. In vitro, animal and human results were extracted from the hits obtained. RESULTS: High cardiac energy demand is sustained by mitochondrial ATP production, and abnormal mitochondrial function has been associated with several lifestyle- and aging-related pathologies in the developed world such as diabetes, non-alcoholic fatty liver disease (NAFLD) and kidney diseases, that in turn can lead to cardiac injury. In order to delay cardiac mitochondrial dysfunction in the context of cardiovascular risk, regular physical activity has been shown to improve mitochondrial parameters and myocardial tolerance to ischemia-reperfusion (IR). Furthermore, pharmacological interventions can prevent the risk of CVDs. Therapeutic agents that can target mitochondria, decreasing ROS production and improve its function have been intensively researched. One example is the mitochondria-targeted antioxidant MitoQ10, which already showed beneficial effects in hypertensive rat models. Carvedilol or antidiabetic drugs also showed protective effects by preventing cardiac mitochondrial oxidative damage. CONCLUSION: This review highlights the role of mitochondrial dysfunction in CVDs, also show-casing several approaches that act by improving mitochondrial function in the heart, contributing to decrease some of the risk factors associated with CVDs.