Triglyceride Accumulation In Tissues Research Articles

Liver-specific overexpression of SIRT3 enhances oxidative metabolism, but does not impact metabolic defects induced by high fat feeding in mice

The mitochondrial enzyme SIRT3 is an NAD+-dependent deacetylase important in cell metabolism, and a decline in its protein expression or activity has been linked with insulin resistance in obesity, ageing and type 2 diabetes. While studies in SIRT3 knockout mice have dramatically improved our understanding of the function of SIRT3, the impact of increasing SIRT3 levels remains under-examined. In this study we investigated the effects of liver-specific SIRT3 overexpression in mice on mitochondrial function and metabolic profile in both isolated hepatocytes and in vivo. Primary hepatocytes overexpressing SIRT3 displayed increased oxygen consumption and a reduction in triglyceride accumulation. In mice with hepatic SIRT3 overexpression, increased fasting β-hydroxybutyrate levels were observed, coupled with an increase in oxygen consumption in isolated mitochondria and increased substrate utilization in liver homogenates. However, metabolic profiling of mice exposed to either chow or high-fat diet revealed no effect of hepatic SIRT3 overexpression on glucose tolerance, body composition or tissue triglyceride accumulation. These findings suggest limited whole-body benefit of increasing hepatic SIRT3 during the development of diet-induced insulin resistance.

The role of glucose, insulin and NEFA in regulating tissue triglyceride accumulation: Substrate cooperation in adipose tissue versus substrate competition in skeletal muscle

Background and aimsMetabolic factors initiating adipose tissue expansion and ectopic triglyceride accumulation are not completely understood. We aimed to investigate the independent role of circulating glucose, NEFA and insulin on glucose and NEFA uptake, and lipogenesis in skeletal muscle and subcutaneous adipose tissue (SCAT). Methods and resultsTwenty-two pigs were stratified according to four protocols: 1) and 2) low NEFA + high insulin ± high glucose (hyperinsulinaemia–hyperglycaemia or hyperinsulinaemia–euglycaemia), 3) high NEFA + low insulin (fasting), 4) low NEFA + low insulin (nicotinic acid). Positron emission tomography with [18F]fluoro-2-deoxyglucose and [11C]acetate, was combined with [14C]acetate and [U-13C]palmitate enrichment techniques to assess glucose and lipid metabolism.Hyperinsulinaemia increased glucose extraction, whilst hyperglycaemia enhanced glucose uptake in skeletal muscle and SCAT. In SCAT, during hyperglycaemia, elevated glucose uptake was accompanied by greater [U-13C]palmitate-TG enrichment compared to the other groups, and by a 39% increase in de novo lipogenesis (DNL) compared to baseline, consistent with a 70% increment in plasma lipogenic index. Conversely, in skeletal muscle, [U-13C]palmitate-TG enrichment was higher after prolonged fasting. ConclusionsOur data show the necessary role of hyperglycaemia–hyperinsulinaemia vs euglycaemia–hyperinsulinaemia in promoting expansion of TG stores in SCAT, by the consensual elevation in plasma NEFA and glucose uptake and DNL. In contrast, skeletal muscle NEFA uptake for TG synthesis is primarily driven by circulating NEFA levels. These results suggest that a) prolonged fasting or dietary regimens enhancing lipolysis might promote muscle steatosis, and b) the control of glucose levels, in association with adequate energy balance, might contribute to weight loss.

PCSK9 Induces CD36 Degradation and Affects Long-Chain Fatty Acid Uptake and Triglyceride Metabolism in Adipocytes and in Mouse Liver.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of the low-density lipoprotein receptor thereby elevating plasma low-density lipoprotein cholesterol levels and the risk of coronary heart disease. Thus, the use of PCSK9 inhibitors holds great promise to prevent heart disease. Previous work found that PCSK9 is involved in triglyceride metabolism, independently of its action on low-density lipoprotein receptor, and that other yet unidentified receptors could mediate this effect. Therefore, we assessed whether PCSK9 enhances the degradation of CD36, a major receptor involved in transport of long-chain fatty acids and triglyceride storage. Overexpressed or recombinant PCSK9 induced CD36 degradation in cell lines and primary adipocytes and reduced the uptake of the palmitate analog Bodipy FL C16 and oxidized low-density lipoprotein in 3T3-L1 adipocytes and hepatic HepG2 cells, respectively. Surface plasmon resonance, coimmunoprecipitation, confocal immunofluorescence microscopy, and protein degradation pathway inhibitors revealed that PCSK9 directly interacts with CD36 and targets the receptor to lysosomes through a mechanism involving the proteasome. Importantly, the level of CD36 protein was increased by >3-fold upon small interfering RNA knockdown of endogenous PCSK9 in hepatic cells and similarly increased in the liver and visceral adipose tissue of Pcsk9(-/-) mice. In Pcsk9(-/-) mice, increased hepatic CD36 was correlated with an amplified uptake of fatty acid and accumulation of triglycerides and lipid droplets. Our results demonstrate an important role of PCSK9 in modulating the function of CD36 and triglyceride metabolism. PCSK9-mediated CD36 degradation may serve to limit fatty acid uptake and triglyceride accumulation in tissues, such as the liver.

8,8-Dimethyldihydroberberine with improved bioavailability and oral efficacy on obese and diabetic mouse models

The clinical use of the natural alkaloid berberine (BBR) as an antidiabetic reagent is limited by its poor bioavailability. Our previous work demonstrated that dihydroberberine (dhBBR) has enhanced bioavailability and in vivo efficacy compared with berberine. Here we synthesized the 8,8-dimethyldihydroberberine (Di-Me) with improved stability, and bioavailability over dhBBR. Similar to BBR and dhBBR, Di-Me inhibited mitochondria respiration, increased AMP:ATP ratio, activated AMPK and stimulated glucose uptake in L6 myotubes. In diet-induced obese (DIO) mice, Di-Me counteracted the increased adiposity, tissue triglyceride accumulation and insulin resistance, and improved glucose tolerance at a dosage of 15 mg/kg. Administered to db/ db mice with a dosage of 50 mg/kg, Di-Me effectively reduced random fed and fasting blood glucose, improved glucose tolerance, alleviated insulin resistance and reduced plasma triglycerides, with better efficacy than dhBBR at the same dosage. Our work highlights the importance of dihydroberberine analogs as potential therapeutic reagents for type 2 diabetes treatment.

Prolonged exposure to high dietary lipids is not associated with lipotoxicity in heart failure

Previous studies have reported that elevated myocardial lipids in a model of mild-to-moderate heart failure increased mitochondrial function, but did not alter left ventricular function. Whether more prolonged exposure to high dietary lipids would promote a lipotoxic phenotype in mitochondrial and myocardial contractile function has not been determined. We tested the hypothesis that prolonged exposure to high dietary lipids, following coronary artery ligation, would preserve myocardial and mitochondrial function in heart failure. Rats underwent ligation or sham surgery and were fed normal (10% kcal fat) (SHAM, HF) or high fat diet (60% kcal saturated fat) (SHAM+FAT, HF+FAT) for sixteen weeks. Although high dietary fat was accompanied by myocardial tissue triglyceride accumulation (SHAM 1.47 ± 0.14; SHAM+FAT 2.32 ± 0.14; HF 1.34 ± 0.14; HF+FAT 2.21 ± 0.20 μmol/gww), fractional shortening was increased 16% in SHAM+FAT and 28% in HF+FAT compared to SHAM and HF, respectively. Despite increased medium-chain acyl-CoA dehydrogenase (MCAD) activity in interfibrillar mitochondria (IFM) of both SHAM+FAT and HF+FAT, dietary lipids also were associated with decreased state 3 respiration using palmitoylcarnitine (SHAM 369 ± 14; SHAM+FAT 307 ± 23; HF 354 ± 13; HF+FAT 366 ± 18 nAO min − 1 mg − 1 ) in SHAM+FAT compared to SHAM and HF+FAT. State 3 respiration in IFM also was decreased in SHAM+FAT relative to SHAM using succinate and DHQ. In conclusion, high dietary lipids promoted myocardial lipid accumulation, but were not accompanied by alterations in myocardial contractile function typically associated with lipotoxicity. In normal animals, high dietary fat decreased mitochondrial respiration, but also increased MCAD activity. These studies support the concept that high fat feeding can modify multiple cellular pathways that differentially affect mitochondrial function under normal and pathological conditions.

Berberine and Its More Biologically Available Derivative, Dihydroberberine, Inhibit Mitochondrial Respiratory Complex I

OBJECTIVE—Berberine (BBR) activates AMP-activated protein kinase (AMPK) and improves insulin sensitivity in rodent models of insulin resistance. We investigated the mechanism of activation of AMPK by BBR and explored whether derivatization of BBR could improve its in vivo efficacy. RESEARCH DESIGN AND METHODS—AMPK phosphorylation was examined in L6 myotubes and LKB1−/− cells, with or without the Ca2+/calmodulin-dependent protein kinase kinase (CAMKK) inhibitor STO-609. Oxygen consumption was measured in L6 myotubes and isolated muscle mitochondria. The effect of a BBR derivative, dihydroberberine (dhBBR), on adiposity and glucose metabolism was examined in rodents fed a high-fat diet. RESULTS—We have made the following novel observations: 1) BBR dose-dependently inhibited respiration in L6 myotubes and muscle mitochondria, through a specific effect on respiratory complex I, similar to that observed with metformin and rosiglitazone; 2) activation of AMPK by BBR did not rely on the activity of either LKB1 or CAMKKβ, consistent with major regulation at the level of the AMPK phosphatase; and 3) a novel BBR derivative, dhBBR, was identified that displayed improved in vivo efficacy in terms of counteracting increased adiposity, tissue triglyceride accumulation, and insulin resistance in high-fat–fed rodents. This effect is likely due to enhanced oral bioavailability. CONCLUSIONS—Complex I of the respiratory chain represents a major target for compounds that improve whole-body insulin sensitivity through increased AMPK activity. The identification of a novel derivative of BBR with improved in vivo efficacy highlights the potential importance of BBR as a novel therapy for the treatment of type 2 diabetes.

The lipogenic enzymes DGAT1, FAS, and LPL in adipose tissue: effects of obesity, insulin resistance, and TZD treatment

Acyl-coenzyme A:diacylglycerol transferase (DGAT), fatty acid synthetase (FAS), and LPL are three enzymes important in adipose tissue triglyceride accumulation. To study the relationship of DGAT1, FAS, and LPL with insulin, we examined adipose mRNA expression of these genes in subjects with a wide range of insulin sensitivity (SI). DGAT1 and FAS (but not LPL) expression were strongly correlated with SI. In addition, the expression of DGAT1 and FAS (but not LPL) were higher in normal glucose-tolerant subjects compared with subjects with impaired glucose tolerance (IGT) (P < 0.005). To study the effects of insulin sensitizers, subjects with IGT were treated with pioglitazone or metformin for 10 weeks, and lipogenic enzymes were measured in adipose tissue. After pioglitazone treatment, DGAT1 expression was increased by 33 +/- 10% (P < 0.05) and FAS expression increased by 63 +/- 8% (P < 0.05); however, LPL expression was not altered. DGAT1, FAS, and LPL mRNA expression were not significantly changed after metformin treatment. The treatment of mice with rosiglitazone also resulted in an increase in adipose expression of DGAT1 by 2- to 3-fold, as did the treatment of 3T3 F442A adipocytes in vitro with thiazolidinediones. These data support a more global concept suggesting that adipose lipid storage functions to prevent peripheral lipotoxicity.

Plasma calcitonin gene-related peptide is increased prior to obesity, and sensory nerve desensitization by capsaicin improves oral glucose tolerance in obese Zucker rats

It has earlier been demonstrated that capsaicin-induced desensitization improves insulin sensitivity in normal rats. However, whether increased capsaicin-sensitive nerve activity precedes the onset of insulin resistance in diet-induced obesity--and therefore might be involved in the pathophysiology--is not known. Further, it is of relevance to investigate whether capsaicin desensitization improves glycaemic control even in obese individuals and we therefore chose the obese Zucker rats to test this. Plasma levels of calcitonin gene-related peptide (CGRP; a marker of sensory nerve activity) was assessed in 8-week-old Zucker rats. To investigate whether capsaicin desensitization (100 mg/kg at 9 weeks of age) would also ameliorate glycaemia in this non-diabetic model, we assessed oral glucose tolerance at 7 weeks after capsaicin. It was found that plasma CGRP levels were elevated in obese Zucker rats prior to the onset of obesity (16.1+/-3.4 pmol/l in pre-obese Zucker rats vs 6.9+/-1.1 pmol/l in lean littermates; P = 0.015) despite similar body weights. Furthermore, capsaicin desensitization reduced both fasting blood glucose (4.3+/-0.2 mmol/l vs 5.1+/-0.2 mmol/l in controls; P = 0.050) as well as the mean blood glucose level during an oral glucose tolerance test (OGTT) (6.8+/-0.3 mmol/l vs 8.6+/-0.5 mmol/l in control obese rats; P = 0.024) whereas the plasma insulin levels during the OGTT were unchanged. However this did not lead to an improvement in insulin resistance or to a reduction of tissue triglyceride accumulation in muscle or liver. We concluded that capsaicin-induced sensory nerve desensitization improves glucose tolerance in Zucker rats. Since, in this study, plasma CGRP levels, a marker of sensory nerve activity, were increased in the pre-obese rats, our data support the hypothesis that increased activity of sensory nerves precedes the development of obesity and insulin resistance in Zucker rats.

Increased diacylglycerol acyltransferase activity is associated with triglyceride accumulation in tissues of diet-induced insulin-resistant hyperlipidemic hamsters

Over-accumulation of triglyceride (TG) in insulin-sensitive tissues is associated with the development of insulin resistance. We investigated whether enhanced de novo lipogenesis via diacylglycerol acyltransferase (DGAT) may contribute to the over-accumulation of TG in various tissues (liver, adipose, muscle, and intestine) using 2 well-characterized hyperlipidemic, insulin-resistant hamster models. In general, a marked increase in TG accumulation was noted in most tissues. Interestingly, the increase in TG accumulation corresponded to an increase in microsomal DGAT activity which ranged from 114% to 575% in all of the examined tissues (n = 7 per group). To delineate the mechanism for the increase in DGAT activity, we measured the expression of DGAT-1 and DGAT-2 messenger RNA by relative reverse transcriptase polymerase chain reaction (RT-PCR). In general, DGAT gene expression changed with DGAT-1 changing the most in the liver and adipose tissue, whereas DGAT-2 showed responses mainly in muscle and intestine. The increases in messenger RNA expression were not remarkable (averaging 35%; n = 4 per group) indicating that posttranscriptional mechanism(s) may play a larger role in regulating DGAT activity. In summary, the data suggest that elevated DGAT activity/expression and the subsequent increase in de novo lipogenesis could in part induce the insulin-resistant state.

An Increase in Peroxisomal Fatty Acid Oxidation Is Not Sufficient to Prevent Tissue Lipid Accumulation in hHTg Rats

We observed earlier that increased skeletal muscle lipid content in the hereditary hypertriglyceridemic (hHTg) rat is accompanied by a decline in plasma leptin. Leptin has recently been shown to enhance peripheral insulin sensitivity by decreasing the tissue triglyceride accumulation, possibly through regulation of fatty acid oxidation and lipogenesis. Thus, to test the hypothesis that insulin resistance and increased skeletal muscle lipid accumulation in hHTg rats are due to a defect in lipid catabolism, we measured mitochondrial and peroxisomal fatty acid oxidation and malonyl-CoA and acetyl-CoA carboxylase-2 content in skeletal muscles of these animals. In addition, we investigated possible molecular mechanisms responsible for the lower leptin levels in hHTg rats by measuring leptin and leptin-receptor (Ob-Ra) mRNA levels. We found the following: (1) in spite of a higher skeletal muscle malonyl-CoA content and an increased sensitivity of carnitine palmitoyltransferase-1 to malonyl-CoA, carnitine palmitoyltransferase-1 activity in muscle of hHTg rats was normal; (2) increased peroxisomal fatty acid oxidation did not seem to be sufficient to prevent the tissue lipid accumulation in these animals; (3) both lower leptin production by white adipose tissue and increased leptin uptake seem to be responsible for lower circulating leptin levels and therefore lower fatty acid catabolism.

Decrease in triglyceride accumulation in tissues by restricted diet and improvement of diabetes in otsuka long-evans tokushima fatty rats, a non—insulin-dependent diabetes model

With respect to the connection between triglyceride (TG) and non-insulin-dependent diabetes mellitus (NIDDM), previous reports have shown that TG accumulation in the liver and muscle is one of the causes of insulin resistance, and TG accumulation in pancreatic islets induces impairment of pancreatic beta-cell function. This experiment examined the relationship between an amelioration of hypertriglyceridemia (HTG), a decrease in TG accumulation in tissues, and an improvement of NIDDM by food restriction. In this experiment using Otsuka Long-Evans Tokushima fatty (OLETF) rats developing NIDDM and Long-Evans Tokushima Otsuka (LETO) rats as controls, sequential changes in body weight and TG content in tissue were measured and biochemical blood tests, an insulin euglycemic clamp test, and histopathologic examination of the pancreas and liver were performed. OLETF rats were allocated to a food-satiated group (satiated) or 30% food-restricted group (restricted). As a result, several findings were more evident in the restricted group than in the satiated group: (1) reductions in body weight and intraabdominal fat weight, decreases in plasma TG, insulin, and glucose levels, a decrease in the TG secretion rate, and an increase in plasma lipoprotein lipase (LPL) activity, (2) decreases in the TG content in the liver, pancreas, and muscle, (3) improvement of the glucose infusion rate (GIR), and (4) a marked reduction of TG accumulation in the liver and pancreatic islets on histopathologic examination. These results indicate that the improved HTG caused a reduction in TG accumulation in the liver and muscle, thereby improving insulin resistance. Moreover, the decrease in TG accumulation in pancreatic islets suggests an improvement of pancreatic beta-cell function.

Relationship between a Decrease in Triglyceride Accumulation in Tissues by Caloric Restriction and an Improvement in Diabetes in OLETF Rats

Relationship between a Decrease in Triglyceride Accumulation in Tissues by Caloric Restriction and an Improvement in Diabetes in OLETF Rats