Obesity progression causes liver steatosis and co-morbidities without apparent cardiac metabolic and functional decline

The goal of this study was to test if obesity progression can be a risk factor to alter cardiac metabolism and function along the time. Male Wistar rats were randomly divided to receive either chow diet (12.0% calories from fat) [C group] or high-fat diet (49.7% calories from fat) plus sucrose in the drinking water (100% from carbohydrate) [H group] for 6, 12 and 24 weeks. The Western diet significantly increased adiposity index of rats in all three experimental periods compared to C group. This was associated with increased plasma levels of insulin, resistin, leptin, glucose, triacylglycerol and decreased adiponectin, however, all variables were stable along the time except insulin and leptin. Plasma free fatty acid was only elevated with 24 weeks treatment. The obesity status resulted in hepatic steatosis progression in H group, while oxidative stress, hepatic inflammatory foci as well as TNF-α and IL-6 mRNA levels were not affected. There are no cardiac performance decline as well as metabolism cardiac changes in H group when compared with C. In conclusion, Western diet induced and promoted obesity, co-morbidities and hepatic steatosis progression while was not associated Nascimento et al. Obesity progression causes liver steatosis and co-morbidities without apparent cardiac metabolic and functional decline 77 with apparent alterations of cardiac metabolism and function. These results suggest that obesity progression seems to affect the organs of distinct ways, and cardiac dysfunction is a question of time.


Introduction
Obesity is defined as abnormal or excessive fat accumulation and has been involved with a strong risk for the development of heart failure (KENCHAIAH et al. 2002;LOEHR et al. 2009). Despite obstructive coronary heart disease is a likely contributor to heart failure in obese subjects, analysis of the Framingham Heart Study (KENCHAIAH et al. 2002) and Atherosclerosis Risk in Communities Study (LOEHR et al. 2009) also demonstrated a significant association between obesity and heart failure, even when analyses were adjusted for myocardial infarction and baseline covariates known to increase the risk of coronary heart disease, including diabetes and hypertension. This suggests that structural and functional alterations of heart among obese individuals are independent of coronary heart disease and reflect pathophysiological alterations in obesity that are both extrinsic and intrinsic to the cardiac tissue.
Overnutrition is one of the most costly challenges for public health. So, understanding the relationship between mechanisms promoting effective calorie storage and adverse metabolic consequences of obesity such as cardiac metabolic dysfunction is emergently necessary. Storage of extra calories in adipose tissue as triglycerides is advantageous, since the ability to sequester lipid effectively inside adipocytes prevents toxic lipid accumulation in other non-adipose tissues, such as muscle, liver and heart, a phenomenon called lipotoxicity (MARRA & SVEGLIATI-BARONI 2018;GHABEN & SCHERER 2019;LOPASCHUK et al. 2007;NISHI et al. 2019;. However, under "metabolically unhealthy obesity" (GHABEN & SCHERER 2019), inappropriate storage of calories increases levels of glucose and lipids in circulation and, consequently, alters cardiac substrate metabolism (WENDE & ABEL et al. 2010). This fact offers a risk for cardiac dysfunction (NISHI et al. 2019;CAROBBIO et al. 2017;D'SOUZA et al. 2016). Although the role of obesity on cardiac metabolism and function is established, the relationship between cardiac metabolism/function with the development of long-term of obesity are not entirely understood. The goal of this study was to test if obesity progression can be a risk factor to alter cardiac metabolism and function along the time.
For inducing obesity progression, we fed Wistar rats with a Western type diet, that mimics food habits of humans living in Western countries, for 6, 12 and 24 weeks. Obesity and co-morbidities were characterized. The cardiac morphology and function were evaluated through echocardiography. Metabolism cardiac was evaluated by measuring enzymes of energy metabolism in cardiac tissue.

Animals and experimental model
Ten-weeks old male Wistar rats (n=90) (São Paulo State University Animal Center -UNESP -Botucatu/SP) were randomly divided to receive either commercial chow diet [C group; 3.77 kcal/g, being 12.0% energy from fat] or high-fat diet [H group; 5.25 kcal/g, being 49.7% energy from fat] for 6, 12 and 24 weeks. The composition of the control and high-fat diet was described in detail in our previous study (LUVIZOTTO et al. 2013). H rats were also given 30% sucrose in drinking water along with the diet, whereas normal drinking water without any supplementation was given to C rats. The diet model was used for mimicking food habits of humans living in Western countries.
Food consumption (g) was measured every day. Rats were housed in individual cages in a temperature-(24 ± 2ºC) and humidity-(55 ± 5%) controlled environment on a 12-12 hour light-dark cycle. The study protocol was approved (CEEA 891-2011) by Botucatu School of Medicine Research Ethics Committee -UNESP and followed the Guide for Care and Use of Experimental Animals.

Cardiac morphology, performance and energy metabolism Analyze of morphology and function
Echocardiographic evaluation was performed using a commercially available echocardiograph (General Electric Medical Systems, Vivid S6, Tirat Carmel, Israel) equipped with a 5 -11.5 MHz multifrequency probe, as described before (NASCIMENTO et al. 2011). The analysed variables were: the left ventricular (LV) end-diastolic dimension (LVDD), posterior wall thickness (PWTd), and anterior wall thickness (AWTd) in diastole were measured at themaximum diastolic dimension. The LV end-systolic dimension (LVSD), posterior wall thickness (PWTs), and anteriorwall thickness (AWTs) in systole were taken at the maximum anterior motion of the posterior wall. The left atrial dimension (LA), aortic dimension (AO) and heart rate (HR) were also measured. Relative wall thickness (RWT) was determined by PWT/LVDD. Left ventricular mass (LVM) was calculated using the following formula:

Statistical analysis
Data are reported as means ± standard deviation. Comparisons between groups were performed using two-way analysis of variance (ANOVA) for independent groups and completed using the post hoc Tukey test. A 5% significance level was adopted. Score of steatosis was presented as median ± semi-range and descriptive statistic was used.

Results and discussion
Final body weight was higher in the H rats than in the C after 12 and 24 weeks (Figure 1). Western diet also up-regulated adiposity index and plasma glucose, triacylglycerol (Figure 1), insulin, leptin and resistin in H group, while down-regulated plasma adiponectin (Table 1), at all experimental time points. Plasma free fatty acid was elevated only at 24 weeks in H group when compared to C ( Figure  1). There was no difference between H and C rats for systolic blood pressure (data not shown).
Absent of steatosis was observed in the livers of rats fed with the control diet in all periods (Score= 0±0), whereas a significant accumulation of fat droplets in the liver was observed in H groups at 6, 12 and 24 weeks (Score: 1±1, 1±1 and 3±1, respectively). Hepatic inflammatory foci were not found in all experimental groups (data not shown); also, TNF--6 mRNA levels, in liver, were not altered in H groups when compared to C (data not shown). The hepatic triacylglycerol accumulation was significantly elevated in H group when compared to C at 6, 12 and 24 weeks ( Figure 2). There were no alteration of TAP, MDA, nitrotirosine and caspase-3 in H group when compared to the C group at all experimental periods ( Figure 2). Together, all data showed there is no hepatic inflammation and injury, suggesting isolated liver steatosis, however, absent of nonalcoholic steatohepatitis and/or advanced liver injury.
H group showed higher left ventricular diastolic dimension than C, while presented lower ratio between left ventricular posterior wall thickness and diastolic dimension, after 12 and 24 weeks of Western diet, suggesting a subtle change in the left ventricular geometry (Table 2). In relation to function, there were no differences in parameters involved with both left ventricular diastolic and systolic function, except cardiac output that increased in H group at 12 and 24 weeks (Table 2). In relation to cardiac metabolic enzymes, Western diet up-regulated both pyruvate dehydrogenase and β-hydroxyacyl coenzyme-A dehydrogenase enzymes in H group, however, just in six week's period. There was no difference to citrate synthase between H and C groups at all experimental moments (Table 3).
Western diet used in this study caused obesity progression in animals, which was confirmed by the elevated adiposity index in association with higher body weight in the H group along the time (Figure 1). Obesity was not related with apparent cardiac metabolic and functional decline, even in the presence of systemic metabolic abnormalities such as hyperglycemia and dyslipidemia (Figure 1) as well as increased leptin, insulin and resistin plasma levels and decreased adiponectin plasma levels (Table 1), and liver steatosis progression. These results provide new insights relating the evolution of obesity and metabolism and cardiac function.     weeks. Data are expressed as means ± SD. Capital letters (6 vs 12 vs 24 weeks); lower letters (C vs H). Different letters indicate significant difference (p<0,05, Two Way ANOVA, post hoc Tukey Test). TG, triacylglycerol; TAP, total antioxindat performance; MDA, malondialdehyde.
Obesity has been associated with cardiac morphology and function abnormalities in a variety of ways (ALPERT et al. 2016;ALPERT et al. 2018). This fact reflects several hemodynamic, neurohormonal and metabolic abnormalities, as well as degree and duration, associated with obesity. In our study, 12 and 24 weeks of obesity were related to higher left ventricular diastolic dimension and increased cardiac output, suggesting a supra-normal left ventricular ejection and subtle change in the left ventricular geometry. Heart rate was mildly elevated in 12 and 24 weeks. Thus, the rise in cardiac output in obese group, in part, should be due the increased heart rate. In other hand, higher left ventricular diastolic dimension could be involved with increased cardiac preload, increasing cardiac output by Frank-Starling mechanism. Another mechanism present in this change is that the excess adipose accumulation in obese individuals leads to an increase in total and central blood volume (ALEXANDER & ALPERT 1998), which in turn predisposes to higher preload and, consequently, elevated cardiac output; also, it explain partially the higher left ventricular diastolic dimension in this study as an eccentric left ventricular hypertrophy that occurs with left ventricular volume overload states. Additionally, the influence of adipose tissue on other aspects of systemic metabolic homeostasis has been appreciated (GHABEN & SCHERER 2019). Adipose tissue expansion by hyperplasia is generally considered healthy and adaptive, preventing toxic lipid accumulation in other tissues, such as liver and heart, and consequently function of the organ. Although liver steatosis has increased along the time in obese group, our results showed no lipid accumulation in myocardium of H animals during all treatment periods (data no shown). In this study, to check the oxidation of fatty acids and glucose by the myocardium metabolic enzymes were also evaluated in cardiac tissue, and was observed that only in short-term of obesity (6 weeks) up-regulated both pyruvate dehydrogenase and β-hydroxyacyl coenzyme-A dehydrogenase; this may be due to the fact that the body is faced with a large supply of fat (hypertriglyceridemia) and glucose (hyperglycemia) to the heart, but there is no relationship with cardiac morphology and function, which was both normal at 6 weeks. To our understanding, chronic obesity might be created a cardiac metabolic homeostasis at 12 and 24 weeks without metabolic dysfunction for promoting cardiac dysfunction. Also, we believe that adipose tissue expansion in our study may have prevented additional systemic metabolic imbalance, buffering additional systemic metabolic dysfunction and, consequently, prevent cardiac disease.
Nonalcoholic fatty liver disease (NAFLD) is a metabolic liver disease characterized by an extensive continuum of liver injury, varying from pure steatosis to nonalcoholic steatohepatitis, fibrosis and cirrhosis, being commonly seen among patients with other metabolic disorders, such as obesity (CLARK et al. 2002;ANGULO 2002). In the last years, two meta-analysis (BORGES-CUNHA et al. 2019;BONCI et al. 2015) demonstrated that NAFLD, especially NASH, associates with adverse structural alterations and cardiac dysfunction, suggesting that liver injury may represent an additional contributor to cardiac alterations. In this study, hepatic steatosis increased along the time, which was not accompanied by inflammation. It was also demonstrated that obesity and steatosis progression was not associated with liver oxidative stress and injury ( Figure 2). Additionally, free fatty acid was mildly elevated in 24 weeks (Figure 1). Taken together, these results suggest that hepatic steatosis may be an adaptive response to the higher plasmatic free fatty acid levels from adipose tissue without promoting additional insults to the heart.
Multiple hemodynamic, neurohormonal and metabolic variables, as well as interact between them, play a key role in the development of cardiac alterations, such as -angiotensin-aldosterone and sympathetic nervous system activation, insulin resistance, hyperleptinemia, and others (ALPERT et al. 2018;. Here, we demonstrated that obesity was associated with comorbidities such as hyperglycemia and hypertriglyceridemia (Figure 1) as well as increased leptin, insulin and resistin plasma levels and decreased adiponectin plasma levels (Table 1). However, these co-morbidities remained stable along the time, except insulin and leptin; both increased in a time-dependent manner in obese ( Table 1). As mentioned above, free fatty acid was also elevated in 24 weeks (Figure 1). Recently, in the same experimental model, our group has demonstrated that both IL-6 and TNF-α were increased in epididymal adipose tissue of 24 weeks obese animals when compared to control as well as short-term of obesity (6 weeks). Also, adipocyte hypertrophy and inflammatory cells as well as Tolllike receptor-4 and NF-κB were only observed at 24 weeks (FRANCISQUETTI et al. 2017). These results allowed us to demonstrate that adipose tissue is influenced by time of obesity, and inflammation of the adipose tissue is associated with adipocyte hypertrophy and TLR-4 activation. Thus, Western diet-associated obesity progression seems to affect the organs of distinct ways. While cardiac alterations are subtle with long-term of obesity demonstrated in this study, adipose tissue has been associated with inflammation as well as insulin resistance and lipolysis, as demonstrated before by our group (FRANCISQUETTI et al. 2017), increasing plasmatic free fatty acid and liver steatosis at 24 weeks. Thus, we believe that cardiac dysfunction is time-manner question.

Conclusion
In conclusion, this present study provided further insights into the western diet-long terminduced obesity progression and its related disease. Chronic exposure to western diet induces obesity and hepatic steatosis progression as well as comorbidities while was not associated with altered myocardial substrate metabolism and apparent cardiac dysfunction along the time.
However, adipose tissue inflammation and liver steatosis are conditions already evident. These results suggest that obesity progression seems to affect the organs of distinct ways, and cardiac dysfunction is a question of time.