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The objective of this paper is to evaluate adaptations in hepatic mitochondrial protein mass, function and efficiency in a rat model of high-fat diet-induced obesity and insulin resistance that displays several correlates to human obesity. Adult male rats were fed a high-fat diet for 7 weeks. Mitochondrial state 3 and state 4 respiratory capacities were measured in liver homogenate and isolated mitochondria by using nicotinamide adenine dinucleotide, flavin adenine dinucleotide and lipid substrates. Mitochondrial efficiency was evaluated by measuring proton leak kinetics. Mitochondrial mass was assessed by ultrastructural observations and citrate synthase (CS) activity measurements. Mitochondrial oxidative damage and antioxidant defence were also considered by measuring lipid peroxidation, aconitase and superoxide dismutase (SOD) specific activity. Whole body metabolic characteristics were obtained by measuring 24-h oxygen consumption (VO2), carbon dioxide production (VCO2), respiratory quotient (RQ) and nonprotein respiratory quotient (NPRQ), using indirect calorimetry with urinary nitrogen analysis. Whole body glucose homeostasis was assessed by measuring plasma insulin and glucose levels after a glucose load. Adult rats fed a high-fat diet for 7 weeks, exhibit not only obesity, insulin resistance and hepatic steatosis, but also reduced respiratory capacity and increased oxidative stress in liver mitochondria. Our present results indicate that alterations in the mitochondrial compartment induced by a high-fat diet are associated with the development of insulin resistance and ectopic fat storage in the liver. Our results thus fit in with the emerging idea that mitochondrial dysfunction can led to the development of metabolic diseases, such as obesity, type 2 diabetes mellitus and nonalcoholic steatohepatitis.

The recovery of body weight after a period of caloric restriction is accompanied by an enhanced efficiency of fat deposition and hyperinsulinemia—which are exacerbated by isocaloric refeeding on a high fat diet rich in saturated and monounsaturated fatty acids (SFA-MUFA), and poor in polyunsaturated fatty acids (PUFA), and associated with a blunting of de novo lipogenesis in adipose tissue and liver. As high fat diets rich in PUFA have been shown to limit the excess fat deposition and improve glucose homeostasis, we investigated here the extent to which de novo lipogenesis in liver and adipose tissues (white and brown), as well as hepatic oxidative stress, are influenced by refeeding on diets rich in PUFA.Design: In rats calorically restricted for 14 days and refed for 14 days on isocaloric amounts of a high fat diet rich in lard (i.e., high SFA- MUFA) or in safflower and linseed oils (rich in PUFA), we investigated energy balance, body composition, glycemic profile, and the regulation of fatty acid synthase (rate- limiting enzyme of de novo lipogenesis) in liver, white and brown adipose tissue. We also evaluated oxidative stress in liver and skeletal muscle and markers of hepatic inflammation.Results: Rats refed the PUFA diet gained less lipids and more proteins compared to rats refed SFA-MUFA diet and showed lower amount of visceral and epididymal white adipose tissue, but increased depots of interscapular brown adipose tissue, with higher expression of the uncoupling protein 1. A significant increase in non- protein respiratory quotient and carbohydrate utilization was found in rats refed PUFA diet. Rats refed PUFA diet showed improved glucose homeostasis, as well as lower triglycerides and cholesterol levels. Fatty acid synthase activity was significantly higher in liver, white and brown adipose tissue, while lipid peroxidation and the degree of inflammation in the liver were significantly lower, in rats refed PUFA diet.Conclusions: When considering the composition of high fat diets for nutritional rehabilitation, the inclusion of PUFA could be useful for improving protein deposition and maintaining glucose homeostasis, while limiting lipid storage in adipose tissue and oxidative stress and inflammation in the liver.

Mitochondria are the main cellular sites devoted to ATP production and lipid oxidation. Therefore, the mitochondrial dysfunction could be an important determinant of cellular fate of circulating lipids, that accumulate in the cytoplasm, if they are not oxidized. The ectopic fat accumulation is associated with the development of insulin resistance, and a link between mitochondrial dysfunction and insulin resistance has been proposed.Recent data on the possible link existing between mitochondrial dysfunction in the liver and diet induced obesity will be summarized, focusing on the three factors that affect the mitochondrial oxidation of metabolic fuels, i.e. organelle number, organelle activity, and energetic efficiency of the mitochondrial machinery in synthesizing ATP. Search in PubMed relevant articles from 2003 to 2014 was conducted, by using query “liver mitochondria and obesity” “hepatic mitochondria and obesity” “liver mitochondria and high fat diet” and “hepatic mitochondria and high fat diet” and including related articles by the same groups.Several works, by using different physiological approaches, have dealt with alteration in mitochondrial function in obesity and diabetes. Most results show that hepatic mitochondrial function is impaired in models of obesity and insulin resistance induced by high-fat or highfructose feeding.Since mitochondria are the main producers of both cellular energy and free radicals, dysfunctional mitochondria could play an important role in the development of insulin resistance and ectopic fat storage in the liver, thus supporting the emerging idea that mitochondrial dysfunction is closely related to the development of obesity, type 2 diabetes mellitus and non-alcoholic steatohepatitis.

Insulin resistance, "a relative impairment in the ability of insulin to exert its effects on glucose, protein and lipid metabolism in target tissues," has many detrimental effects on metabolism and is strongly correlated to deposition of lipids in non-adipose tissues. Mitochondria are the main cellular sites devoted to ATP production and fatty acid oxidation. Therefore, a role for mitochondrial dysfunction in the onset of skeletal muscle insulin resistance has been proposed and many studies have dealt with possible alteration in mitochondrial function in obesity and diabetes, both in humans and animal models. Data reporting evidence of mitochondrial dysfunction in type two diabetes mellitus are numerous, even though the issue that this reduced mitochondrial function is causal in the development of the disease is not yet solved, also because a variety of parameters have been used in the studies carried out on this subject. By assessing the alterations in mitochondrial efficiency as well as the impact of this parameter on metabolic homeostasis of skeletal muscle cells, we have obtained results that allow us to suggest that an increase in mitochondrial efficiency precedes and therefore can contribute to the development of high-fat-induced insulin resistance in skeletal muscle.

To explore the effect of a fructose-rich diet on morphological and functional changes in white adipose tissue (WAT) that could contribute to the development of insulin resistance. Adult sedentary rats were fed a fructose-rich diet for 8 weeks. Glucose tolerance test was carried out together with measurement of plasma triglycerides, non-esterified fatty acids and lipid peroxidation. In subcutaneous abdominal and intra-abdominal WAT, number and size of adipocytes together with cellular insulin sensitivity and lipolytic activity were assessed. Rats fed a fructose-rich diet exhibited a significant increase in plasma insulin, triglycerides, non-esterified fatty acids and lipid peroxidation, together with significantly increased body lipids and epididymal and mesenteric WAT, compared to controls. Mean adipocyte volume in subcutaneous abdominal WAT was significantly lower, while mean adipocyte volume in intra-abdominal WAT was significantly higher, in rats fed a fructose-rich diet compared to controls. A significant increase in larger adipocytes and a significant decrease in smaller adipocytes were found in intra-abdominal WAT in rats fed a fructose-rich diet compared to controls. Insulin’s ability to inhibit lipolysis was blunted in subcutaneous abdominal and intra-abdominal adipocytes from fructose-fed rats. Accordingly, lower p-Akt/Akt ratio was found in WAT in rats fed a fructose-rich diet compared to controls. Long-term consumption of high levels of fructose elicits remarkable morphological and functional modifications, particularly in intra-abdominal WAT, that are highly predictive of obesity and insulin resistance and that contribute to the worsening of metabolic alterations peculiar in a fructose-rich, hypolipidic diet.

To assess hepatic de novo lipogenesis and mitochondrial energetics as well as whole-body energy homeostasis in sedentary rats fed a fructose-rich diet. Male rats of 90 days of age were fed a high-fructose or control diet for 8 weeks. Body composition, energy balance, oxygen consumption, carbon dioxide production, non-protein respiratory quotient, de novo lipogenesis and insulin resistance were measured. Determination of specific activity of hepatic enzymes of de novo lipogenesis, mitochondrial mass, oxidative capacity and degree of coupling, together with parameters of oxidative stress and antioxidant defence, was also carried out. Body energy and lipid content as well as plasma insulin and non-esterified fatty acids were significantly higher in fructose-fed than in control rats. Significantly higher rates of net de novo lipogenesis and activities of hepatic lipogenic enzymes fatty acid synthase and stearoyl CoA desaturase-1 were found in fructose-fed rats compared to controls. Mitochondrial protein mass and degree of coupling were significantly higher in fructose-fed rats compared to controls. Hepatic mitochondria showed oxidative damage, both in the lipid and in the protein component, together with decreased activity of antioxidant defence. Liver mitochondrial compartment is highly affected by fructose feeding. The increased mitochondrial efficiency allows liver cells to burn less substrates to produce ATP for de novo lipogenesis and gluconeogenesis. In addition, increased lipogenesis gives rise to whole body and ectopic lipid deposition, and higher mitochondrial coupling causes mitochondrial oxidative stress.

In the present study, the effect of long-term fructose feeding on skeletal muscle mitochondrial energetics was investigated. Measurements in isolated tissue were coupled with the determination of whole-body energy expenditure and insulin sensitivity. A significant increase in plasma NEFA, as well as in skeletal muscle TAG and ceramide, was found in fructose-fed rats compared with the controls, together with a significantly higher plasma insulin response to a glucose load, while no significant variation in plasma glucose levels was found. Significantly lower RMR values were found in fructose-fed rats starting from week 4 of the dietary treatment. Skeletal muscle mitochondrial mass and degree of coupling were found to be significantly higher in fructose-fed rats compared with the controls. Significantly higher lipid peroxidation was found in fructose-fed rats, together with a significant decrease in superoxide dismutase activity. Phosphorylated Akt levels normalised to plasma insulin levels were significantly lower in fructose-fed rats compared with the controls. In conclusion, a fructose-rich diet has a deep impact on a metabolically relevant tissue such as skeletal muscle. In this tissue, the consequences of high fructose feeding are altered glucose tolerance, elevated mitochondrial biogenesis and increased mitochondrial coupling. This latter modification could have a detrimental metabolic effect by causing oxidative stress and energy sparing that contribute to the high metabolic efficiency of fructose-fed rats.