Circulating isomiRs May Be Superior Biomarkers Compared to Their Corresponding miRNAs: A Pilot Biomarker Study of Using isomiR-Ome to Detect Coronary Calcium-Based Cardiovascular Risk in Patients with NAFLD.

Circulating miRNAs are increasingly being considered as biomarkers in various medical contexts, but the value of analyzing isomiRs (isoforms of canonical miRNA sequences) has not frequently been assessed. Here we hypothesize that an in-depth analysis of the full circulating miRNA landscape could identify specific isomiRs that are stronger biomarkers, compared to their corresponding miRNA, for identifying increased CV risk in patients with non-alcoholic fatty liver disease (NAFLD)-a clinical unmet need. Plasma miRNAs were sequenced with next-generation sequencing (NGS). Liver fat content was measured with magnetic-resonance spectrometry (MRS); CV risk was determined, beyond using traditional biomarkers, by a CT-based measurement of coronary artery calcium (CAC) score and the calculation of a CAC score-based CV-risk percentile (CAC-CV%). This pilot study included n = 13 patients, age > 45 years, with an MRS-measured liver fat content of ≥5% (wt/wt), and free of overt CVD. NGS identified 1103 miRNAs and 404,022 different isomiRs, of which 280 (25%) and 1418 (0.35%), respectively, passed an abundance threshold. Eighteen (sixteen/two) circulating miRNAs correlated positively/negatively, respectively, with CAC-CV%, nine of which also significantly discriminated between high/low CV risk through ROC-AUC analysis. IsomiR-ome analyses uncovered 67 isomiRs highly correlated (R ≥ 0.55) with CAC-CV%. Specific isomiRs of miRNAs 101-3p, 144-3p, 421, and 484 exhibited stronger associations with CAC-CV% compared to their corresponding miRNA. Additionally, while miRNAs 140-3p, 223-3p, 30e-5p, and 342-3p did not correlate with CAC-CV%, specific isomiRs with altered seed sequences exhibited a strong correlation with coronary atherosclerosis burden. Their predicted isomiRs-specific targets were uniquely enriched (compared to their canonical miRNA sequence) in CV Disease (CVD)-related pathways. Two of the isomiRs exhibited discriminative ROC-AUC, and another two showed a correlation with reverse cholesterol transport from cholesterol-loaded macrophages to ApoB-depleted plasma. In summary, we propose a pipeline for exploring circulating isomiR-ome as an approach to uncover novel and strong CVD biomarkers.

CTRP6 rapidly responds to acute nutritional changes, regulating adipose tissue expansion and inflammation in mice.

In chronic obesity, activated adipose tissue proinflammatory cascades are tightly linked to metabolic dysfunction. Yet, close temporal analyses of the responses to obesogenic environment such as high-fat feeding (HFF) in susceptible mouse strains question the causal relationship between inflammation and metabolic dysfunction, and/or raises the possibility that certain inflammatory cascades play adaptive/homeostatic, rather than pathogenic roles. Here, we hypothesized that CTRP6, a C1QTNF family member, may constitute an early responder to acute nutritional changes in adipose tissue, with potential physiological roles. Both 3-days high-fat feeding (3dHFF) and acute obesity reversal [2-wk switch to low-fat diet after 8-wk HFF (8wHFF)] already induced marked changes in whole body fuel utilization. Although adipose tissue expression of classical proinflammatory cytokines (Tnf-α, Ccl2, and Il1b) exhibited no, or only minor, change, C1qtnf6 uniquely increased, and decreased, in response to 3dHFF and acute obesity reversal, respectively. CTRP6 knockout (KO) mouse embryonic fibroblasts (MEFs) exhibited increased adipogenic gene expression (Pparg, Fabp4, and Adipoq) and markedly reduced inflammatory genes (Tnf-α, Ccl2, and Il6) compared with wild-type MEFs, and recombinant CTRP6 induced the opposite gene expression signature, as assessed by RNA sequencing. Consistently, 3dHFF of CTRP6-KO mice induced a greater whole body and adipose tissue weight gain compared with wild-type littermates. Collectively, we propose CTRP6 as a gene that rapidly responds to acute changes in caloric intake, acting in acute overnutrition to induce a "physiological inflammatory response" that limits adipose tissue expansion.NEW & NOTEWORTHY CTRP6 (C1qTNF6), a member of adiponectin gene family, regulates inflammation and metabolism in established obesity. Here, short-term high-fat feeding in mice is shown to increase adipose tissue expression of CTRP6 before changes in the expression of classical inflammatory genes occur. Conversely, CTRP6 expression in adipose tissue decreases early in the course of obesity reversal. Gain- and loss-of-function models suggest CTRP6 as a positive regulator of inflammatory cascades, and a negative regulator of adipogenesis and adipose tissue expansion.

A new implantable tool for repeated assessment of supraventricular electrophysiology and atrial fibrillation susceptibility in freely moving rats.

The complex pathophysiology of atrial fibrillation (AF) is governed by multiple risk factors in ways that are still elusive. Basic electrophysiological properties, including atrial effective refractory period (AERP) and conduction velocity, are major factors determining the susceptibility of the atrial myocardium to AF. Although there is a great need for affordable animal models in this field of research, in vivo rodent studies are limited by technical challenges. Recently, we introduced an implantable system for long-term assessment of AF susceptibility in ambulatory rats. However, technical considerations did not allow us to perform concomitant supraventricular electrophysiology measurements. Here, we designed a novel quadripolar electrode specifically adapted for comprehensive atrial studies in ambulatory rats. Electrodes were fabricated from medical-grade silicone, four platinum-iridium poles, and stainless-steel fixating pins. Initial quality validation was performed ex vivo, followed by implantation in adult rats and repeated electrophysiological studies 1, 4, and 8 wk postimplantation. Capture threshold was stable. Baseline AERP values (38.1 ± 2.3 and 39.5 ± 2.0 using 70-ms and 120-ms S1-S1 cycle lengths, respectively) confirmed the expected absence of rate adaptation in the unanesthetized state and validated our prediction that markedly higher values reported under anesthesia are nonphysiological. Evaluation of AF substrate in parallel with electrophysiological parameters validated our recent finding of a gradual increase in AF susceptibility over time and demonstrated that this phenomenon is associated with an electrical remodeling process characterized by AERP shortening. Our findings indicate that the miniature quadripolar electrode is a potent new tool, which opens a window of opportunities for better utilization of rats in AF research.NEW & NOTEWORTHY Rodents are increasingly used in AF research. However, technical challenges restrict long-term supraventricular electrophysiology studies in these species. Here, we developed an implantable electrode adapted for such studies in the rat. Our findings indicate that this new tool is effective for long-term follow-up of critical parameters such as atrial refractoriness. Obtained data shed light on the normal electrophysiology and on the increased AF susceptibility that develops in rats with implanted atrial electrodes over time.

Modulation of gut microbiota by spent coffee grounds attenuates diet-induced metabolic syndrome in rats.

Coffee brewing produces spent coffee grounds as waste; few studies have investigated the health benefits of these grounds. This study investigated responses to spent coffee grounds in a diet-induced rat model of metabolic syndrome. Male Wistar rats aged 8-9 weeks were fed either corn starch-rich diet or high-carbohydrate, high-fat diet for 16 weeks, which were supplemented with 5% spent coffee grounds during the last 8 weeks. Rats fed non-supplemented diets were used as controls. High-carbohydrate, high-fat diet-fed rats developed metabolic syndrome including abdominal obesity, impaired glucose tolerance, dyslipidemia, and cardiovascular and liver damage. Body weight, abdominal fat, total body fat mass, systolic blood pressure, and concentrations of plasma triglycerides and non-esterified fatty acids were reduced by spent coffee grounds along with improved glucose tolerance and structure and function of heart and liver. Spent coffee grounds increased the diversity of the gut microbiota and decreased the ratio of Firmicutes to Bacteroidetes. Changes in gut microbiota correlated with the reduction in obesity and improvement in glucose tolerance and systolic blood pressure. These findings indicate that intervention with spent coffee grounds may be useful for managing obesity and metabolic syndrome by altering the gut microbiota, thus increasing the value of this food waste.

Attenuation of Metabolic Syndrome by EPA/DHA Ethyl Esters in Testosterone-Deficient Obese Rats.

Inducing testosterone deficiency, as the standard treatment of prostate cancer, may cause metabolic disorders including insulin resistance, dyslipidemia, central obesity, cardiovascular diseases, and type 2 diabetes. This study measured responses to testosterone deficiency in high-carbohydrate, high-fat (H) diet-fed rats. We then tested whether eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) ethyl esters (Omacor) reversed these metabolic changes. Male Wistar rats (8⁻9 weeks old) were divided into eight groups with four groups fed corn starch and four groups fed H diet. For each diet, one group received diet only; one group was orchidectomized; one group was given leuprolide (gonadotrophin-releasing hormone agonist, 2 mg/kg every 4th week); and the last group was treated with leuprolide and their diet was supplemented with 3% Omacor for the last eight weeks. The protocol was for 16 weeks. Leuprolide worsened metabolic syndrome symptoms and cardiovascular function, and orchidectomy produced greater responses. In H fed leuprolide-treated rats, Omacor decreased systolic blood pressure and left ventricular diastolic stiffness, reduced infiltration of inflammatory cells and collagen deposition in the heart, and reduced lipid accumulation and inflammatory cell infiltration without improving liver damage. These results suggest that Omacor has potential to attenuate metabolic complications in prostate cancer patients with induced testosterone deprivation.

Coffee Pulp, a By-Product of Coffee Production, Modulates Gut Microbiota and Improves Metabolic Syndrome in High-Carbohydrate, High-Fat Diet-Fed Rats.

Waste from food production can be re-purposed as raw material for usable products to decrease industrial waste. Coffee pulp is 29% of the dry weight of coffee cherries and contains caffeine, chlorogenic acid, trigonelline, diterpenes and fibre. We investigated the attenuation of signs of metabolic syndrome induced by high-carbohydrate, high-fat diet in rats by dietary supplementation with 5% freeze-dried coffee pulp for the final 8 weeks of a 16-week protocol. Coffee pulp decreased body weight, feed efficiency and abdominal fat; normalised systolic blood pressure, left ventricular diastolic stiffness, and plasma concentrations of triglycerides and non-esterified fatty acids; and improved glucose tolerance in rats fed high-carbohydrate, high-fat diet. Further, the gut microbiota was modulated with high-carbohydrate, high-fat diet and coffee pulp supplementation and 14 physiological parameters were correlated with the changes in bacterial community structures. This study suggested that coffee pulp, as a waste from the coffee industry, is useful as a functional food for improving obesity-associated metabolic, cardiovascular and liver structure and function, and gut microbiota.

Adaptation of fuel selection to acute decrease in voluntary energy expenditure is governed by dietary macronutrient composition in mice.

In humans, exercise-induced thermogenesis is a markedly variable component of total energy expenditure, which had been acutely affected worldwide by COVID-19 pandemic-related lockdowns. We hypothesized that dietary macronutrient composition may affect metabolic adaptation/fuel selection in response to an acute decrease in voluntary activity. Using mice fed short-term high-fat diet (HFD) compared to low-fat diet (LFD)-fed mice, we evaluated whole-body fuel utilization by metabolic cages before and 3 days after omitting a voluntary running wheel in the cage. Short-term (24-48 h) HFD was sufficient to increase energy intake, fat oxidation, and decrease carbohydrate oxidation. Running wheel omission did not change energy intake, but resulted in a significant 50% decrease in total activity and a ~20% in energy expenditure in the active phase (night-time), compared to the period with wheel, irrespective of the dietary composition, resulting in significant weight gain. Yet, while in LFD wheel omission significantly decreased active phase fat oxidation, thereby trending to increase respiratory exchange ratio (RER), in HFD it diminished active phase carbohydrate oxidation. In conclusion, acute decrease in voluntary activity resulted in positive energy balance in mice on both diets, and decreased oxidation of the minor energy (macronutrient) fuel source, demonstrating that dietary macronutrient composition determines fuel utilization choices under conditions of acute changes in energetic demand.

Chlorogenic acid attenuates high-carbohydrate, high-fat diet-induced cardiovascular, liver, and metabolic changes in rats.

Chlorogenic acid as a constituent of coffee is consumed regularly in the human diet. Chlorogenic acid intake has been associated with decreased risk of cardiovascular disease and type 2 diabetes. We hypothesized that chlorogenic acid would improve cardiovascular, liver, and metabolic responses in a rat model of metabolic syndrome induced by a high-carbohydrate, high-fat diet. Male Wistar rats (8-9 weeks old, 335 ±â€¯2 g, n = 48) were divided into 4 groups and fed with corn starch diet (16 weeks); corn starch diet with chlorogenic acid in food for the last 8 weeks; high-carbohydrate, high-fat diet (16 weeks); or high-carbohydrate, high-fat diet with chlorogenic acid (~100 mg/kg/d) in food for the last 8 weeks. In high-carbohydrate, high-fat diet-fed rats, chlorogenic acid reduced energy intake and food efficiency to reduce visceral fat, especially retroperitoneal fat, and abdominal circumference; reversed the elevated systolic blood pressure; and attenuated left ventricular diastolic stiffness with reduced collagen deposition and infiltration of inflammatory cells in the left ventricle. Chlorogenic acid decreased inflammation and fat deposition in the liver along with reduced plasma liver enzyme activities of obese rats but did not change the plasma lipid profile. Chlorogenic acid increased diversity of gut microbiota, which may improve overall metabolism in the body. Thus, chronic dietary chlorogenic acid attenuated diet-induced inflammation as well as cardiovascular, liver, and metabolic changes, suggesting that chlorogenic acid has potential for further clinical evaluation.