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Metabolic dysfunction-associated fatty liver disease (MAFLD) represents a growing cause of mortality and morbidity and encompasses a spectrum of liver pathologies. While dozens of preclinical models have been developed to recapitulate stages of MAFLD, few achieve fibrosis using an experimental design that mimics human pathogenesis. We sought to clarify whether the combination of thermoneutral (TN) housing and consumption of a classical Western diet (WD) would accelerate the onset and progression of MAFLD. Male and female C57Bl/6J mice were fed a nutrient-matched low-fat control or Western diet (WD) for 16 weeks. Mice were housed with littermates at either standard temperature (TS; 22°C) or thermoneutral-like conditions (TN; ~29°C). Male but not female mice housed at TN and fed a WD were significantly heavier than TS -housed control animals. WD-fed mice housed under TN conditions had lower levels of circulating glucose compared to TS mice; however, there were select but minimal differences in other circulating markers. While WD-fed TN males had higher liver enzyme and higher liver triglyceride levels, no differences in markers of liver injury or hepatic lipid accumulation were observed in females. Housing temperature had little effect on histopathological scoring of MAFLD progression in males; however, while female mice retained a level of protection, WD-TN conditions trended toward a worsened hepatic phenotype, which was associated with higher macrophage transcript expression and content. Our results indicate that interventions coupling TN housing and WD-induced MAFLD should be longer than 16 weeks to accelerate hepatic steatosis and increase inflammation in both sexes of mice.

Objective: Nobiletin is a dietary flavonoid that improves insulin resistance and atherosclerosis in mice with metabolic dysfunction. Dysregulation of intestinal lipoprotein metabolism contributes to atherogenesis. The objective of the study was to determine if nobiletin targets the intestine to improve metabolic dysregulation in both male and female mice. Approach and Results: Triglyceride-rich lipoprotein (TRL) secretion, intracellular triglyceride kinetics, and intestinal morphology were determined in male and female LDL (low-density lipoprotein) receptor knockout ( Ldlr –/– ), and male wild-type mice fed a standard laboratory diet or high-fat, high-cholesterol (HFHC) diet ± nobiletin using an olive oil gavage, radiotracers, and electron microscopy. Nobiletin attenuated postprandial TRL levels in plasma and enhanced TRL clearance. Nobiletin reduced fasting jejunal triglyceride accumulation through accelerated TRL secretion and lower jejunal fatty acid synthesis with no impact on fatty acid oxidation. Fasting-refeeding experiments revealed that nobiletin led to higher levels of phosphorylated AKT (protein kinase B) and FoxO1 (forkhead box O1) and normal Srebf1c expression indicating increased insulin sensitivity. Intestinal length and weight were diminished by HFHC feeding and restored by nobiletin. Both fasting and postprandial plasma GLP-1 (glucagon-like peptide-1; and likely GLP-2) were elevated in response to nobiletin. Treatment with a GLP-2 receptor antagonist, GLP-2(3-33), reduced villus length in HFHC-fed mice but did not impact TRL secretion in any diet group. In contrast to males, nobiletin did not improve postprandial lipid parameters in female mice. Conclusions: Nobiletin opposed the effects of the HFHC diet by normalizing intestinal de novo lipogenesis through improved insulin sensitivity. Nobiletin prevents postprandial lipemia because the enhanced TRL clearance more than compensates for increased TRL secretion.

Ischemic heart disease is the leading cause of death in the United States, Canada, and worldwide. Severe disease is characterized by coronary artery occlusion, loss of blood flow to the myocardium, and necrosis of tissue, with subsequent remodeling of the heart wall, including fibrotic scarring. The current study aims to demonstrate the efficacy of quantitating infarct size via two-dimensional (2-D) echocardiographic akinetic length and four-dimensional (4-D) echocardiographic infarct volume and surface area as in vivo analysis techniques. We further describe and evaluate a new surface area strain analysis technique for estimating myocardial infarction (MI) size after ischemic injury. Experimental MI was induced in mice via left coronary artery ligation. Ejection fraction and infarct size were measured through 2-D and 4-D echocardiography. Infarct size established via histology was compared with ultrasound-based metrics via linear regression analysis. Two-dimensional echocardiographic akinetic length (r = 0.76, P = 0.03), 4-D echocardiographic infarct volume (r = 0.85, P = 0.008), and surface area (r = 0.90, P = 0.002) correlate well with histology. Although both 2-D and 4-D echocardiography were reliable measurement techniques to assess infarct, 4-D analysis is superior in assessing asymmetry of the left ventricle and the infarct. Strain analysis performed on 4-D data also provides additional infarct sizing techniques, which correlate with histology (surface strain: r = 0.94, P < 0.001, transmural thickness: r = 0.76, P = 0.001). Two-dimensional echocardiographic akinetic length, 4-D echocardiography ultrasound, and strain provide effective in vivo methods for measuring fibrotic scarring after MI. NEW & NOTEWORTHY Our study supports that both 2-D and 4-D echocardiographic analysis techniques are reliable in quantifying infarct size though 4-D ultrasound provides a more holistic image of LV function and structure, especially after myocardial infarction. Furthermore, 4-D strain analysis correctly identifies infarct size and regional LV dysfunction after MI. Therefore, these techniques can improve functional insight into the impact of pharmacological interventions on the pathophysiology of cardiac disease.

ObjectiveMetabolic dysfunction-associated fatty liver disease (MAFLD) represents a growing cause of mortality and morbidity and encompasses a spectrum of liver pathologies. Potential therapeutic targets have been identified and are currently being pre-clinically and clinically tested. However, while dozens of preclinical models have been developed to recapitulate various stages of MAFLD, few achieve fibrosis using an experimental design that mimics human pathogenesis. We sought to clarify whether the combination of thermoneutral (TN) housing and consumption of a classical Western diet (WD) would accelerate the onset of MAFLD and progression in male and female mice.MethodsMale and female C57Bl/6J mice were fed a nutrient-matched low-fat control or Western diet (41% Kcal from fat, 43% carbohydrate and 0.2% cholesterol; WD) starting at ∼12 wk of age for a further 16 wk. Mice were divided and housed with littermates at either standard temperature (TS; 22°C) or thermoneutral conditions (TN; ∼29°C). Mice underwent tests for glucose tolerance, insulin sensitivity and body composition, as well as intestinal permeability. Following tissue harvest, circulating and liver markers of hepatic disease progression toward steatosis and fibrosis were determined.ResultsWhile male mice housed at TNand fed a WD were significantly heavier than TS-housed control animals, no other differences in body weight or composition were observed. WD-fed females housed under TNconditions had higher circulating LDL-cholesterol; however, there were no other significant differences between TNand TS-housing in circulating or hepatic lipid levels. While WD-fed TNmales had higher ALT levels, no other differences in markers of liver injury or disease progression were observed. Moreover, females housed at TNconditions and fed a WD remained significantly protected against the induction of fibrosis compared to male counterparts. Interestingly, sex-specific differences were observed in markers of glucose and insulin tolerance, where TNhousing and WD feeding resulted in hyperglycemia and impaired insulin responsiveness in both sexes, but glucose intolerance only in male mice.ConclusionsWhile TNhousing has been demonstrated to exacerbate high fat-induced hepatic steatosis and inflammation in male and female mice, coupling TNhousing with a WD for 16 wk was not sufficient to augment fatty liver progression in male or female mice.HighlightsThermoneutral housing and Western diet feeding does not progress to NASHFemale mice are not more susceptible to obesity induced fatty liver under these conditionsTemperature and diet had sex-specific effects on glucose tolerance and insulin sensitivity

Elevated circulating dipeptidyl-peptidase 4 is a biomarker for liver disease, but its involvement in gluconeogenesis and in metabolic-associated fatty liver disease (MAFLD) progression remains unclear. Here we identified that DPP4 in hepatocytes but not Tie2+ endothelial cells regulates the local bioactivity of incretin hormones and gluconeogenesis. However, the complete absence of DPP4 (Dpp4-/-) in aged mice with metabolic syndrome accelerates liver fibrosis without altering dyslipidemia and steatosis. Analysis of transcripts from the livers of whole body Dpp4-/- displayed enrichment for inflammasome, p53, and senescence programs compared to littermate controls. High-fat high-cholesterol (HFHC)-feeding decreased Dpp4 expression in F4/80+ cells, with only minor changes in immune signaling. Moreover, in a lean mouse model of severe non-alcoholic fatty liver disease (NAFLD), phosphatidylethanolamine N-methyltransferase (Pemt -/-) mice fed with HFHC diet, we observed a 4-fold increase in circulating DPP4, disassociating its release from obesity. Lastly, we evaluated DPP4 levels in patients with hepatitis C infection with dysglycemia (HOMA-IR2) who underwent direct antiviral treatment (with or without ribavirin). DPP4 protein levels decreased with viral clearance, and DPP4 activity levels were reduced at longer-term follow-up in ribavirin-treated patients, although metabolic factors did not improve. These data suggest elevations in DPP4 during HCV infection are not primarily regulated by metabolic disturbances.

Introduction: The small intestine receives hormonal and neuronal stimuli to ensure sufficient nutrient absorption to fuel the body. Insulin resistance promotes aberrant dietary fat delivery into circulation, increasing the risk of cardiovascular disease. The mechanisms underlying the loss of controlled dietary fat delivery during metabolic disease are unclear. We aimed to characterize the metabolic pathways engaged in the small intestine in the transition from fasting to refeeding states in both male and female mice. Methods: Eight- to twelve-week-old male and female mice fasted for 24 hours. Half of the mice were euthanized in the fasted state, and the remaining half were euthanized after a 4-hour refeeding period (Teklad Global 19% Protein Extruded Diet). Gene expression and pathway analyses in jejunal samples were determined using Nanostring. Results: In male and female mice, refeeding significantly upregulated pathways in amino acid synthesis, mitochondrial respiration, and proto-oncogene Myc signalling compared to fasting. Refeeding significantly downregulated glycolysis, and both mitogen-activated MAPK and PI3K signalling pathways compared to fasting. Interestingly, in males refeeding significantly upregulated AMPK, mTOR, and autophagy, whereas conversely in female mice, refeeding downregulated these pathways. Surprisingly, fatty acid synthesis and fatty acid oxidation pathways were unchanged by the nutritional state in males, but in females, both pathways were significantly downregulated with refeeding compared to fasting. In female mice, refeeding significantly reduced small intestinal length compared to fasting, whereas intestinal length was unchanged in male mice. Conclusions: Metabolic pathways well-characterized in the liver are often mechanistically extrapolated to the gut. We have begun to uncover the jejunal gene expression changes regulated by nutrient availability in male and female mice. Identifying how high-fat feeding dysregulates these pathways will help further elucidate the mechanism underlying the over-production of intestinally-derived lipoproteins in metabolic disease.

The small intestine is a highly adaptable organ serving as both a barrier to the external environment and a conduit for nutrient absorption. Enterocytes package dietary triglycerides (TG) into chylomicrons for transport into circulation; the remaining TGs are stored in cytosolic lipid droplets (CLDs). The current study aimed to characterize the impact of diet composition on intestinal lipid handling in male and female wild-type mice. Mice were continued on their grain-based diet (GBD) and switched to either a high-fat, high cholesterol Western-style diet (WD) or a ketogenic diet (KD) for 3 or 5 weeks. KD-fed mice displayed significantly higher plasma TG levels in response to an olive oil gavage than WD- and GBD-fed mice; TG levels were ~2-fold higher in male KD-fed mice than female KD-fed mice. Poloxamer-407 experiments revealed enhanced intestinal-TG secretion rates in male mice fed a KD upon olive oil gavage, whereas secretion rates were unchanged in female mice. Surprisingly, jejunal CLD size and TG mass after oil gavage were similar among the groups. At fasting, TG mass was significantly higher in the jejunum of male KD-fed mice and the duodenum of female KD-fed mice, providing increased substrate for chylomicron formation. In addition to greater fasting intestinal TG stores, KD-fed male mice displayed longer small intestinal lengths, while female mice displayed markedly longer jejunal villi lengths. After 5 weeks of diet, 12 h fasting-2 h refeeding experiments revealed jejunal TG levels were similar between diet groups in male mice; however, in female mice, jejunal TG mass was significantly higher in KD-fed mice compared to GBD- and WD-fed mice. These experiments reveal that KD feeding promotes distinct morphological and functional changes to the murine small intestine compared to the WD diet. Moreover, changes to intestinal lipid handling in response to carbohydrate and protein restriction manifest differently in male and female mice.