Maria Pia Mollica

From chronic overnutrition to insulin resistance: the role of fat-storing capacity and inflammation.

AIMS We analyze how the inflammatory state in adipose tissue caused by a condition of chronically positive energy balance can lead to insulin resistance first in adipose tissue, then in all insulin-sensitive tissues. DATA SYNTHESIS Chronic nutrient overload causes an increase in adipose depots that, if adipose tissue expandability is low, are characterized by an increased presence of hypertrophic adipocytes. This adipocyte hypertrophy is a possible stress condition for the endoplasmic reticulum (ER) that would lead to a proinflammatory state in adipose tissue. In this condition, ER stress would activate metabolic pathways that trigger insulin resistance, release of macrophage chemoattractant proteins, and in chronic inflammation, the death of the hypertrophic adipocyte. The infiltrated macrophages in turn release inflammatory proteins causing further recruitment of macrophages to adipose tissue and the release of inflammatory cytokines. Following these events, insulin resistance becomes extended to all adipose tissue. Insulin-resistant adipocytes, characterized by low liposynthetic capacity and high lipolytic capacity, cause increased release of free fatty acids (FFA). FFA released by lipolitic adipocytes may also activate Toll-like receptors 4 and then chemokines and cytokines release amplifying insulin resistance, lipolysis and inflammation in all adipose tissue. Moreover, increased circulating FFA levels, reduced circulating adiponectin levels and leptin resistance lead to decreased lipid oxidation in non-adipose tissues, thereby triggering ectopic accumulation of lipids, lipotoxicity and insulin resistance. CONCLUSION All the conditions that increase circulating fatty acids and cause lipid overloading (obesity, lipoatrophy, lipodystrophy, catabolic states, etc.) induce a lipotoxic state in non-adipose tissues that gives rise to insulin resistance.

Skeletal muscle oxidative capacity in rats fed high-fat diet

OBJECTIVE: To investigate whether young rats respond to high-fat feeding through changes in energy efficiency and fuel partitioning at the level of skeletal muscle, to avoid obesity development. In addition, to establish whether the two mitochondrial subpopulations, which exist in skeletal muscle, ie subsarcolemmal and intermyofibrillar, are differently affected by high-fat feeding.DESIGN: Weaning rats were fed a low-fat or a high-fat diet for 15 days.MEASUREMENTS: Energy balance and lipid partitioning in the whole animal. State 3 and state 4 oxygen consumption rates in whole skeletal muscle homogenate. State 3 and state 4 oxygen consumption rates, membrane potential and uncoupling effect of palmitate in subsarcolemmal and intermyofibrillar mitochondria from skeletal muscle.RESULTS: Rats fed a high-fat diet showed an increased whole body lipid utilization. Skeletal muscle NAD-linked and lipid oxidative capacity significantly increased at the whole-tissue level, due to an increase in lipid oxidative capacity in subsarcolemmal and intermyofibrillar mitochondria and in NAD-linked activity only in intermyofibrillar ones. In addition, rats fed a high-fat diet showed an increase in the uncoupling effect of palmitate in both the mitochondrial populations.CONCLUSIONS: In young rats fed a high-fat diet, skeletal muscle contributes to enhanced whole body lipid oxidation through an increased mitochondrial capacity to use lipids as metabolic fuels, associated with a decrease in energy coupling.

Skeletal muscle subsarcolemmal mitochondrial dysfunction in high-fat fed rats exhibiting impaired glucose homeostasis

Objective:To investigate whether changes in body energy balance induced by long-term high-fat feeding in adult rats could be associated with modifications in energetic behaviour and oxidative stress of skeletal muscle subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial populations.Design:Adult rats were fed low-fat or high-fat diet for 7 weeks.Measurements:Body energy balance and composition analysis together with plasma insulin and glucose level determination in the whole animal. Oxidative capacity, basal and induced proton leaks as well as aconitase and superoxide dismutase activities in SS and IMF mitochondria from skeletal muscle.Results:High-fat fed rats exhibit increased body lipid content, as well as hyperinsulinemia, hyperglycaemia and higher plasma non-esterified fatty acids. In addition, SS mitochondria display lower respiratory capacity and a different behaviour of SS and IMF mitochondria is found in the prevention from oxidative damage.Conclusions:A deleterious consequence of decreased oxidative capacity in SS mitochondria from rats fed high-fat diet would be a reduced utilization of energy substrates, especially fatty acids, which may lead to intracellular triglyceride accumulation, lipotoxicity and insulin resistance development. Our results thus reveal a possible role for SS mitochondria in the impairment of glucose homeostasis induced by high-fat diet.

From chronic overfeeding to hepatic injury: Role of endoplasmic reticulum stress and inflammation

We analyse how chronic overfeeding, by increasing circulating fatty acids, might lead to inflammation, insulin resistance (IR) and injury in the liver. Chronic overfeeding causes an increase in adipose tissue depots and is characterised by an increased presence of hypertrophic adipocytes when adipose tissue expandability is inadequate. Adipocyte hypertrophy is a possible stress condition for the endoplasmic reticulum (ER), which will activate inflammatory and apoptotic pathways and cause IR in adipose tissue. Insulin-resistant adipocytes, being more lipolytic and less liposynthetic, induce an increase in circulating free fatty acids. Moreover, the strongly compromised secretion/function of the adipocyte hormones, adiponectin and leptin, decreases lipid oxidation, particularly in the liver, causing lipid accumulation, ER stress and IR in hepatocytes. ER stress may lead to reduced very-low-density lipoprotein (VLDL) secretion and increased lipogenic gene expression despite the presence of IR. These events and reduced lipid oxidation may lead to further hepatic lipid accumulation. When the triglyceride storage capacity of hepatocytes is exceeded, hepatic injury may occur. ER-stressed steatotic hepatocytes activate apoptotic and inflammatory pathways, which trigger IR and the release of chemokines and cytokines, and these, in turn, elicit an increased influx of Kupffer cells (KCs) and hepatic stellate cells (HSCs) around dying hepatocytes. Soluble mediators, secreted mainly by ER-stressed steatotic hepatocytes and activated KCs, induce the transdifferentiation of HSCs to myofibroblasts, which secrete fibrogenic cytokines and matrix components that trigger fibrosis. In conclusion, chronic lipid overloading due to inadequate fat-storing capacity of adipose tissue can induce hepatic injury when triglyceride storage capacity of hepatocytes is exceeded.

Fat balance and serum leptin concentrations in normal, hypothyroid, and hyperthyroid rats.

OBJECTIVE: To study the influence of thyroid hormones on the relationship between serum leptin and fat mass, as well as on energy and macronutrient balance.DESIGN: Rats with different thyroid states were obtained by 7 and 15 days of treatment with the antithyroid drug propylthiouracil or with triiodothyronine (T3).MEASUREMENTS: Energy balance, macronutrient balance and serum leptin concentrations.RESULTS: In hypothyroid rats we found a decrease in metabolizable energy (ME) intake and energy expenditure together with an increase in lipid gain/lipid intake ratio and a decrease in protein gain/protein intake ratio. Consequently, body lipid percentage significantly increased compared to euthyroid rats. Hyperthyroid rats first increased energy expenditure and later ME intake, so that increased metabolism was balanced by increased intake, and energy gain was similar to that found in euthyroid rats.CONCLUSION: These results indicate that T3 plays a major role in the maintenance of energy and lipid balance. Our results also indicate that an inverse relationship exists between T3 and leptin serum concentrations, and that this relationship is not only the result of changes in body fat stores induced by changed T3 concentrations.

Differences in proton leak kinetics, but not in UCP3 protein content, in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria from fed and fasted rats

We have investigated the effect of 24‐h fasting on basal proton leak and uncoupling protein (UCP) 3 expression at the protein level in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria. In fed rats, the two mitochondrial populations displayed different proton leak, but the same protein content of UCP3. In addition, 24‐h fasting, both at 24 and 29°C, induced an increase in proton leak only in subsarcolemmal mitochondria, while UCP3 content increased in both the populations. From the present data, it appears that UCP3 does not control the basal proton leak of skeletal muscle mitochondria.

Skeletal muscle mitochondrial efficiency and uncoupling protein 3 in overeating rats with increased thermogenesis.

Abstract. To establish whether changes in skeletal muscle mitochondrial efficiency contribute to increased energy expenditure and decreased metabolic efficiency of overeating rats with increased thermogenesis, we measured basal proton leak, fatty acid-induced uncoupling and uncoupling protein 3 (UCP3) content in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria. Intermyofibrillar, but not subsarcolemmal, mitochondria from rats with increased thermogenesis exhibited a lower proton leak compared with controls. In both mitochondrial populations from rats with increased thermogenesis, fatty acid-induced uncoupling was increased significantly and a small recoupling effect of GDP was detected. In addition, intermyofibrillar and subsarcolemmal mitochondria from rats with increased thermogenesis showed higher UCP3 contents than controls. These results point out that metabolic efficiency in subsarcolemmal and intermyofibrillar mitochondria from rats with increased thermogenesis is differently regulated. In fact, in intermyofibrillar mitochondria both basal proton leak and fatty acid-induced uncoupling are altered, while in subsarcolemmal mitochondria only fatty acid-induced uncoupling increases. Both mitochondrial populations in skeletal muscle cells from rats with increased thermogenesis display an increased fatty acid-induced uncoupling and UCP3 content, which could contribute to avoiding obesity.

Acetyl-L-carnitine treatment stimulates oxygen consumption and biosynthetic function in perfused liver of young and old rats

Abstract: The effect of treatment with acetyl-L-carnitine on hepatic mitochondrial respiration and biosynthetic function in perfused liver from young (90 days) and old (22-24 months) rats was studied. Rats were given a 1.5% (w/v) solution of acetyl-L-carnitine in their drinking water for 1 month and oxygen consumption together with the rate of gluconeogenesis, urea synthesis, and ketogenesis with and without added substrates were measured in perfused liver. Mitochondrial oxygen consumption was also assessed in liver homogenate and isolated mitochondria to determine the maximal capacity for oxidative phosphorylation. Acetyl-L-carnitine treatment almost completely restored the age-dependent decline in oxygen consumption, gluconeogenesis, urea synthesis, and ketogenesis found in perfused liver of old rats to the levels found in young rats. In addition, acetyl-L-carnitine treatment increased oxygen consumption and biosynthetic function in perfused liver from young rats. After acetyl-L-carnitine treatment, we found detectable 3-oxoacyl-CoA-transferase activity associated with a consumption of ketone bodies in young and old rats. Finally, oxygen consumption measured in homogenate and isolated mitochondria did not change with age and acetyl-L-carnitine treatment. Our results show that in perfused liver, acetyl-L-carnitine treatment slows the age-associated decline in mitochondrial respiration and biosynthetic function. In addition, treatment of young rats with acetyl-L-carnitine has a stimulating effect on liver metabolism, probably through an increase in ATP production.

Polyphenol-rich virgin olive oil reduces insulin resistance and liver inflammation and improves mitochondrial dysfunction in high-fat diet fed rats.

Scope: Virgin olive oil is an essential component of the Mediterranean diet. Its antioxidant and anti‐inflammatory properties are mainly linked to phenolic contents. This study aims to evaluate the beneficial effects of a polyphenol‐rich virgin olive oil (HPCOO) or olive oil without polyphenols (WPOO) in rats fed high‐fat diet (HFD). Methods and results: Male Sprague‐Dawley rats were divided into four groups based on the different types of diet: (I) standard diet (STD); (II) HFD; (III) HFD containing WPOO, and (IV) HFD containing HPCOO. HPCOO and WPOO induced a significant improvement of HFD‐induced impaired glucose homeostasis (by hyperglycemia, altered oral glucose tolerance, and HOMA‐IR) and inflammatory status modulating pro‐ and anti‐inflammatory cytokines (TNF‐α, IL‐1, and IL‐10) and adipokines. Moreover, HPCOO and less extensively WPOO, limited HFD‐induced liver oxidative and nitrosative stress and increased hepatic fatty acid oxidation. To study mitochondrial performance, oxidative capacity and energy efficiency were also evaluated in isolated liver mitochondria. HPCOO, but not WPOO, reduced H2O2 release and aconitase activity by decreasing degree of coupling, which plays a major role in the control of mitochondrial reactive oxygen species emission. Conclusion: HPCOO limits HFD‐induced insulin resistance, inflammation, and hepatic oxidative stress, preventing nonalcoholic fatty liver disease progression.

Modulation of hepatic mitochondrial energy efficiency with age

This study was designed to examine the effect of youth-adulthood transition on hepatic mitochondrial energy efficiency. The changes in basal and palmitate-induced proton leak, which contribute to mitochondrial efficiency, were evaluated in mitochondria isolated from the liver of young and adult rats. Alterations in mitochondrial cytochrome oxidase and aconitase specific activities, and in adenine nucleotide translocator content were also assessed. There was no difference in basal proton leak or thermodynamic coupling and efficiency of oxidative phosphorylation in liver mitochondria between the two rat groups. On the other hand, palmitate-induced proton leak increased significantly in adult rats. The function of this uncoupling could be avoidance of elevated formation of reactive oxygen species, which are known to accelerate ageing.