Fructose liquid and solid formulations differently affect gut integrity, microbiota composition and related liver toxicity: a comparative in vivo study.

Despite clinical findings suggesting that the form (liquid versus solid) of the sugars may significantly affect the development of metabolic diseases, no experimental data are available on the impact of their formulations on gut microbiota, integrity and hepatic outcomes. In the present sudy, C57Bl/6j mice were fed a standard diet plus water (SD), a standard diet plus 60% fructose syrup (L-Fr) or a 60% fructose solid diet plus water (S-Fr) for 12 weeks. Gut microbiota was characterized through 16S rRNA phylogenetic profiling and shotgun sequencing of microbial genes in ileum content and related volatilome profiling. Fructose feeding led to alterations of the gut microbiota depending on the fructose formulation, with increased colonization by Clostridium, Oscillospira and Clostridiales phyla in the S-Fr group and Bacteroides, Lactobacillus, Lachnospiraceae and Dorea in the L-Fr. S-Fr evoked the highest accumulation of advanced glycation end products and barrier injury in the ileum intestinal mucosa. These effects were associated to a stronger activation of the lipopolysaccharide-dependent proinflammatory TLR4/NLRP3 inflammasome pathway in the liver of S-Fr mice than of L-Fr mice. In contrast, L-Fr intake induced higher levels of hepatosteatosis and markers of fibrosis than S-Fr. Fructose-induced ex novo lipogenesis with production of SCFA and MCFA was confirmed by metagenomic analysis. These results suggest that consumption of fructose under different forms, liquid or solid, may differently affect gut microbiota, thus leading to impairment in intestinal mucosa integrity and liver homeostasis.

Effects of vitamin D on insulin resistance and myosteatosis in diet-induced obese mice.

Epidemiological studies pointed out to a strong association between vitamin D deficiency and type 2 diabetes prevalence. However, the role of vitamin D supplementation in the skeletal muscle, a tissue that play a crucial role in the maintenance of glucose homeostasis, has been scarcely investigated so far. On this basis, this study aimed to evaluate the effect of vitamin D supplementation in a murine model of diet-induced insulin resistance with particular attention to the effects evoked on the skeletal muscle. Male C57BL/6J mice (n = 40) were fed with a control or a High Fat-High Sugar (HFHS) diet for 4 months. Subsets of animals were treated for 2 months with vitamin D (7 µg·kg-1, i.p. three times/week). HFHS diet induced body weight increase, hyperglycemia and impaired glucose tolerance. HFHS animals showed an impaired insulin signaling and a marked fat accumulation in the skeletal muscle. Vitamin D reduced body weight and improved systemic glucose tolerance. In addition, vitamin D restored the impaired muscle insulin signaling and reverted myosteatosis evoked by the diet. These effects were associated to decreased activation of NF-κB and lower levels of TNF-alpha. Consistently, a significantly decreased activation of the SCAP/SREBP lipogenic pathway and lower levels of CML protein adducts and RAGE expression were observed in skeletal muscle of animals treated with vitamin D. Collectively, these data indicate that vitamin D-induced selective inhibition of signaling pathways (including NF-κB, SCAP/SREBP and CML/RAGE cascades) within the skeletal muscle significantly contributed to the beneficial effects of vitamin D supplementation against diet-induced metabolic derangements.

A lake-centric geospatial database to guide research and inform management decisions in an Arctic watershed in northern Alaska experiencing climate and land-use changes.

Lakes are dominant and diverse landscape features in the Arctic, but conventional land cover classification schemes typically map them as a single uniform class. Here, we present a detailed lake-centric geospatial database for an Arctic watershed in northern Alaska. We developed a GIS dataset consisting of 4362 lakes that provides information on lake morphometry, hydrologic connectivity, surface area dynamics, surrounding terrestrial ecotypes, and other important conditions describing Arctic lakes. Analyzing the geospatial database relative to fish and bird survey data shows relations to lake depth and hydrologic connectivity, which are being used to guide research and aid in the management of aquatic resources in the National Petroleum Reserve in Alaska. Further development of similar geospatial databases is needed to better understand and plan for the impacts of ongoing climate and land-use changes occurring across lake-rich landscapes in the Arctic.

Chronic administration of saturated fats and fructose differently affect SREBP activity resulting in different modulation of Nrf2 and Nlrp3 inflammasome pathways in mice liver.

The overconsumption of both saturated fats and fructose in the modern society has been related to the development of nonalcoholic fatty liver disease (NAFLD). However, the specific contribution of individual dietary components on the progression of NAFLD to nonalcoholic steatohepatitis (NASH) has been poorly investigated. Therefore, the aim of our study was to investigate the dissimilar effects of these two dietary components on selected proinflammatory and antioxidant pathways in the liver of C57BL/6 mice fed a standard (SD), a 45% saturated fat (HFAT) or a 60% fructose (HFRT) diet for 12 weeks. HFAT diet evoked systemic metabolic alterations and overweight, not observed in HFRT mice. However, HFRT mice had a greater hepatic triglyceride deposition with increased ratio of triacylglycerols containing the palmitic acid compared to HFAT, as assessed by liquid chromatography-mass spectrometry analysis. This effect is due to the higher activation of the SCAP/SREBP1c lipogenic pathway by HFRT feeding. In addition, we found inhibition of Keap1/Nrf2 antioxidant signaling and more robust stimulation of the Nlrp3 inflammasome pathway in the livers of HFRT-fed mice when compared with HFAT-fed mice, which is consistent with the recent finding that palmitate and SREBP1c are implicated in hepatic oxidative stress and inflammation. These effects were associated with increased hepatic inflammation, as confirmed by high expression of markers of leukocyte infiltration in the HFRT group. Thus, we hypothesize an amplifying loop among lipogenesis, palmitate, Nrf2 and Nlrp3 that leads to a higher risk of NAFLD progression to NASH in a high-fructose diet compared to a high-saturated fat intake.

Effects of chronic sugar consumption on lipid accumulation and autophagy in the skeletal muscle.

PURPOSE: In recent years, the increasing consumption of soft drinks containing high-fructose corn syrup or sucrose has caused a rise in fructose intake, which has been related to the epidemic of metabolic diseases. As fructose and glucose intake varies in parallel, it is still unclear what the effects of the increased consumption of the two single sugars are. In the present study, the impact of chronic consumption of glucose or fructose on skeletal muscle of healthy mice was investigated. METHODS: C57BL/6J male mice received water (C), 15 % fructose (ChF) or 15 % glucose (ChG) to drink for up to 7 months. Lipid metabolism and markers of inflammation and autophagy were assessed in gastrocnemius muscle. RESULTS: Increased body weight and gastrocnemius muscle mass, as well as circulating glucose, insulin, and lipid plasma levels were observed in sugar-drinking mice. Although triglycerides increased in the gastrocnemius muscle of both ChF and ChG mice (+32 and +26 %, vs C, respectively), intramyocellular lipids accumulated to a significantly greater extent in ChF than in ChG animals (ChF +10 % vs ChG). Such perturbations were associated with increased muscle interleukin-6 levels (threefold of C) and with the activation of autophagy, as demonstrated by the overexpression of LC3B-II (ChF, threefold and ChG, twofold of C) and beclin-1 (ChF, sevenfold and ChG, tenfold of C). CONCLUSIONS: The present results suggest that intramyocellular lipids and the pro-inflammatory signaling could contribute to the onset of insulin resistance and lead to the induction of autophagy, which could be an adaptive response to lipotoxicity.

Empagliflozin Protects against Diet-Induced NLRP-3 Inflammasome Activation and Lipid Accumulation.

The aim of this study was to evaluate the effects of chronic treatment with empagliflozin, a potent and selective sodium glucose cotransporter-2 inhibitor, in a murine model of diet-induced obesity and insulin resistance, focusing on drug effects on body weight reduction and nucleotide-binding domain, leucine-rich repeat containing protein (NLRP)-3 inflammasome activation, which have never been investigated to date. Male C57BL/6 mice were fed control or a high fat-high sugar (HFHS) diet for 4 months. Over the last 2 months, subsets of animals were treated with empagliflozin (1-10 mg/kg) added to the diet. Empagliflozin evoked body weight reduction (P < 0.001 for the highest dose) and positive effects on fasting glycemia and homeostasis model assessment of insulin resistance. In addition, the drug was able to reduce renal tubular damage and liver triglycerides level in a dose-dependent manner. Interestingly, empagliflozin also decreased cardiac lipid accumulation. Moreover, diet-induced activation of NLRP-3 in kidney and liver (not observed in the heart) was dose-dependently attenuated by empagliflozin. Our results clearly demonstrate the ability of empagliflozin to counteract the deleterious effects evoked by chronic exposure to HFHS diet. Most notably, empagliflozin treatment was associated with NLRP-3 inflammasome signaling modulation, suggesting that this inhibition may contribute to the drug therapeutic effects.

High-fructose intake as risk factor for neurodegeneration: Key role for carboxy methyllysine accumulation in mice hippocampal neurons.

Several studies indicate the involvement of advanced glycation end-products (AGEs) in neurodegenerative diseases. Moreover, the rising consumption of fructose in industrialized countries has been related to cognitive impairment, but the impact of fructose-derived AGEs on hippocampus has never been investigated. The present study aimed to evaluate in the hippocampus of C57Bl/6 mice fed a standard (SD) or a 60% fructose (HFRT) diet for 12 weeks the production of the most studied AGEs, carboxy methyllysine (CML), focusing on the role of the glutathione-dependent enzyme glyoxalase (Glo-1), the main AGEs-detoxifying system, in relation to early signs of neuronal impairment. HFRT diet evoked CML accumulation in the cell body of pyramidal neurons, followed by RAGE/NFkB signaling activation. A widespread reactive gliosis and altered mitochondrial respiratory complexes activity have been evidenced in HFRT hippocampi, paralleled by oxidative stress increase due to impaired activity of Nrf2 signaling. In addition, a translocation of Glo-1 from axons toward cell body of pyramidal neurons has been observed in HFRT mice, in relation to CML accumulation. Despite increased expression of dimeric Glo-1, its enzymatic activity was not upregulated in HFRT hippocampi, due to reduced glutathione availability, thus failing to prevent CML accumulation. The prevention of CML production by administration of the specific inhibitor pyridoxamine was able to prevent all the fructose-induced hippocampal alterations. In conclusion, a high-fructose consumption, through CML accumulation and Glo-1 impairment, induces in the hippocampus the same molecular and metabolic alterations observed in early phases of neurodegenerative diseases, and can thus represent a risk factor for their onset.

Maladaptive Modulations of NLRP3 Inflammasome and Cardioprotective Pathways Are Involved in Diet-Induced Exacerbation of Myocardial Ischemia/Reperfusion Injury in Mice.

Excessive fatty acids and sugars intake is known to affect the development of cardiovascular diseases, including myocardial infarction. However, the underlying mechanisms are ill defined. Here we investigated the balance between prosurvival and detrimental pathways within the heart of C57Bl/6 male mice fed a standard diet (SD) or a high-fat high-fructose diet (HFHF) for 12 weeks and exposed to cardiac ex vivo ischemia/reperfusion (IR) injury. Dietary manipulation evokes a maladaptive response in heart mice, as demonstrated by the shift of myosin heavy chain isoform content from α to ß, the increased expression of the Nlrp3 inflammasome and markers of oxidative metabolism, and the downregulation of the hypoxia inducible factor- (HIF-)2α and members of the Reperfusion Injury Salvage Kinases (RISK) pathway. When exposed to IR, HFHF mice hearts showed greater infarct size and lactic dehydrogenase release in comparison with SD mice. These effects were associated with an exacerbated overexpression of Nlrp3 inflammasome, resulting in marked caspase-1 activation and a compromised activation of the cardioprotective RISK/HIF-2α pathways. The common mechanisms of damage here reported lead to a better understanding of the cross-talk among prosurvival and detrimental pathways leading to the development of cardiovascular disorders associated with metabolic diseases.

Pharmacological Inhibition of NLRP3 Inflammasome Attenuates Myocardial Ischemia/Reperfusion Injury by Activation of RISK and Mitochondrial Pathways.

Although the nucleotide-binding oligomerization domain- (NOD-) like receptor pyrin domain containing 3 (NLRP3) inflammasome has been recently detected in the heart, its role in cardiac ischemia/reperfusion (IR) is still controversial. Here, we investigate whether a pharmacological modulation of NLRP3 inflammasome exerted protective effects in an ex vivo model of IR injury. Isolated hearts from male Wistar rats (5-6 months old) underwent ischemia (30 min) followed by reperfusion (20 or 60 min) with and without pretreatment with the recently synthetized NLRP3 inflammasome inhibitor INF4E (50 µM, 20 min before ischemia). INF4E exerted protection against myocardial IR, shown by a significant reduction in infarct size and lactate dehydrogenase release and improvement in postischemic left ventricular pressure. The formation of the NLRP3 inflammasome complex was induced by myocardial IR and attenuated by INF4E in a time-dependent way. Interestingly, the hearts of the INF4E-pretreated animals displayed a marked improvement of the protective RISK pathway and this effect was associated increase in expression of markers of mitochondrial oxidative phosphorylation. Our results demonstrate for the first time that INF4E protected against the IR-induced myocardial injury and dysfunction, by a mechanism that involves inhibition of the NLRP3 inflammasome, resulting in the activation of the prosurvival RISK pathway and improvement in mitochondrial function.

Targeting the NLRP3 Inflammasome to Reduce Diet-Induced Metabolic Abnormalities in Mice.

Although the molecular links underlying the causative relationship between chronic low-grade inflammation and insulin resistance are not completely understood, compelling evidence suggests a pivotal role of the nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) inflammasome. Here we tested the hypothesis that either a selective pharmacological inhibition or a genetic downregulation of the NLRP3 inflammasome results in reduction of the diet-induced metabolic alterations. Male C57/BL6 wild-type mice and NLRP3-/- littermates were fed control diet or high-fat, high-fructose diet (HD). A subgroup of HD-fed wild-type mice was treated with the NLRP3 inflammasome inhibitor BAY 11-7082 (3 mg/kg intraperitoneally [IP]). HD feeding increased plasma and hepatic lipids and impaired glucose homeostasis and renal function. Renal and hepatic injury was associated with robust increases in profibrogenic markers, while only minimal fibrosis was recorded. None of these metabolic abnormalities were detected in HD-fed NLRP3-/- mice, and they were dramatically reduced in HD-mice treated with the NLRP3 inflammasome inhibitor. BAY 11-7082 also attenuated the diet-induced increase in NLRP3 inflammasome expression, resulting in inhibition of caspase-1 activation and interleukin (IL)-1ß and IL-18 production (in liver and kidney). Interestingly, BAY 11-7082, but not gene silencing, inhibited nuclear factor (NF)-κB nuclear translocation. Overall, these results demonstrate that the selective pharmacological modulation of the NLRP3 inflammasome attenuates the metabolic abnormalities and the related organ injury/dysfunction caused by chronic exposure to HD, with effects similar to those obtained by NLRP3 gene silencing.

Dynamics of a recovering Arctic bird population: the importance of climate, density dependence, and site quality.

Intrinsic and extrinsic factors affect vital rates and population-level processes, and understanding these factors is paramount to devising successful management plans for wildlife species. For example, birds time migration in response, in part, to local and broadscale climate fluctuations to initiate breeding upon arrival to nesting territories, and prolonged inclement weather early in the breeding season can inhibit egg-laying and reduce productivity. Also, density-dependent regulation occurs in raptor populations, as territory size is related to resource availability. Arctic Peregrine Falcons (Falco peregrinus tundrius; hereafter Arctic peregrine) have a limited and northern breeding distribution, including the Colville River Special Area (CRSA) in the National Petroleum Reserve-Alaska, USA. We quantified influences of climate, topography, nest productivity, prey habitat, density dependence, and interspecific competition affecting Arctic peregrines in the CRSA by applying the Dail-Madsen model to estimate abundance and vital rates of adults on nesting cliffs from 1981 through 2002. Arctic peregrine abundance increased throughout the 1980s, which spanned the population's recovery from DDT-induced reproductive failure, until exhibiting a stationary trend in the 1990s. Apparent survival rate (i.e., emigration; death) was negatively correlated with the number of adult Arctic peregrines on the cliff the previous year, suggesting effects of density-dependent population regulation. Apparent survival and arrival rates (i.e., immigration; recruitment) were higher during years with earlier snowmelt and milder winters, and apparent survival was positively correlated with nesting season maximum daily temperature. Arrival rate was positively correlated with average Arctic peregrine productivity along a cliff segment from the previous year and initial abundance was positively correlated with cliff height. Higher cliffs with documented higher productivity (presumably indicative of higher-quality habitat), are a priority for continued protection from potential nearby development and disturbance to minimize population-level impacts. Climate change. may affect Arctic peregrines in multiple ways, including through access to more snow-free nest sites and a lengthened breeding season that may increase likelihood of nest success. Our work provides insight into factors affecting a population during and after recovery, and demonstrates how the Dail-Madsen model can be used for any unmarked population with multiple years of abundance data collected through repeated surveys.

Accumulation of advanced glycation end-products and activation of the SCAP/SREBP Lipogenetic pathway occur in diet-induced obese mouse skeletal muscle.

Aim of this study was to investigate whether advanced glycation end-products (AGEs) accumulate in skeletal myofibers of two different animal models of diabesity and whether this accumulation could be associated to myosteatosis. Male C57Bl/6j mice and leptin-deficient ob/ob mice were divided into three groups and underwent 15 weeks of dietary manipulation: standard diet-fed C57 group (C57, n = 10), high-fat high-sugar diet-fed C57 group (HFHS, n = 10), and standard diet-fed ob/ob group (OB/OB, n = 8). HFHS mice and OB/OB mice developed glycometabolic abnormalities in association with decreased mass of the gastrocnemius muscle, fast-to-slow transition of muscle fibers, and lipid accumulation (that occurred preferentially in slow compared to fast fibers). Moreover, we found in muscle fibers of HFHS and OB/OB mice accumulation of AGEs that was preferential for the lipid-accumulating cells, increased expression of the lipogenic pathway SCAP/SREBP, and co-localisation between AGEs and SCAP-(hyper)expressing cells (suggestive for SCAP glycosylation). The increased expression of the SCAP/SREBP lipogenic pathway in muscle fibers is a possible mechanism underlying lipid accumulation and linking myosteatosis to muscle fiber atrophy and fast-to-slow transition that occur in response to diabesity.

Variability in myosteatosis and insulin resistance induced by high-fat diet in mouse skeletal muscles.

Nutrient overload leads to impaired muscle oxidative capacity and insulin sensitivity. However, comparative analyses of the effects of dietary manipulation on skeletal muscles with different fiber composition are lacking. This study aimed to investigate the selective adaptations in the soleus and tibialis anterior muscles evoked by administration of high-fat diet for 12 weeks in 10 mice (HFD mice) compared to 10 animals fed with a normal chow diet (control mice). Mice fed with the HFD diet exhibited hyperlipidemia, hyperinsulinemia, hyperglycemia, and lower exercise capacity in comparison to control mice. In control mice, soleus fibers showed higher lipid content than tibialis anterior fibers. In contrast, the lipid content was similar between the two muscles in HFD mice. Significant differences in markers of muscle mitochondrial production and/or activity as well as of lipid synthesis were detected between HFD mice and control mice, especially in the tibialis anterior. Moreover, translocation of GLUT-4 transporter to the plasma membrane and activation of the insulin signaling pathway were markedly inhibited in the tibialis and slightly reduced in the soleus of HFD mice compared to control mice. Overall, these results show that adaptive responses to dietary manipulation occur in a muscle-specific pattern.

Advanced glycation end products promote hepatosteatosis by interfering with SCAP-SREBP pathway in fructose-drinking mice.

Clinical studies have linked the increased consumption of fructose to the development of obesity, dyslipidemia, and impaired glucose tolerance, and a role in hepatosteatosis development is presumed. Fructose can undergo a nonenzymatic reaction from which advanced glycation end products (AGEs) are derived, leading to the formation of dysfunctional, fructosylated proteins; however, the in vivo formation of AGEs from fructose is still less known than that from glucose. In the present study C57Bl/6J mice received 15% (wt/vol) fructose (FRT) or 15% (wt/vol) glucose (GLC) in water to drink for 30 wk, resembling human habit to consume sugary drinks. At the end of the protocol both FRT- and GLC-drinking mice had increased fasting glycemia, glucose intolerance, altered plasma lipid profile, and marked hepatosteatosis. FRT mice had higher hepatic triglycerides deposition than GLC, paralleled by a greater increased expression and activity of the sterol regulatory element-binding protein 1 (SREBP1), the transcription factor responsible for the de novo lipogenesis, and of its activating protein SCAP. LC-MS analysis showed a different pattern of AGE production in liver tissue between FRT and GLC mice, with larger amount of carboxymethyl lysine (CML) generated by fructose. Double immunofluorescence and coimmunoprecipitation analysis revealed an interaction between CML and SCAP that could lead to prolonged activation of SREBP1. Overall, the high levels of CML and activation of SCAP/SREBP pathway associated to high fructose exposure here reported may suggest a key role of this signaling pathway in mediating fructose-induced lipogenesis.