Denis van Beurden

Prolonged Fasting Identifies Skeletal Muscle Mitochondrial Dysfunction as Consequence Rather Than Cause of Human Insulin Resistance

OBJECTIVE Type 2 diabetes and insulin resistance have been associated with mitochondrial dysfunction, but it is debated whether this is a primary factor in the pathogenesis of the disease. To test the concept that mitochondrial dysfunction is secondary to the development of insulin resistance, we employed the unique model of prolonged fasting in humans. Prolonged fasting is a physiologic condition in which muscular insulin resistance develops in the presence of increased free fatty acid (FFA) levels, increased fat oxidation and low glucose and insulin levels. It is therefore anticipated that skeletal muscle mitochondrial function is maintained to accommodate increased fat oxidation unless factors secondary to insulin resistance exert negative effects on mitochondrial function. RESEARCH DESIGN AND METHODS While in a respiration chamber, twelve healthy males were subjected to a 60 h fast and a 60 h normal fed condition in a randomized crossover design. Afterward, insulin sensitivity was assessed using a hyperinsulinemic-euglycemic clamp, and mitochondrial function was quantified ex vivo in permeabilized muscle fibers using high-resolution respirometry. RESULTS Indeed, FFA levels were increased approximately ninefold after 60 h of fasting in healthy male subjects, leading to elevated intramuscular lipid levels and decreased muscular insulin sensitivity. Despite an increase in whole-body fat oxidation, we observed an overall reduction in both coupled state 3 respiration and maximally uncoupled respiration in permeabilized skeletal muscle fibers, which could not be explained by changes in mitochondrial density. CONCLUSIONS These findings confirm that the insulin-resistant state has secondary negative effects on mitochondrial function. Given the low insulin and glucose levels after prolonged fasting, hyperglycemia and insulin action per se can be excluded as underlying mechanisms, pointing toward elevated plasma FFA and/or intramuscular fat accumulation as possible causes for the observed reduction in mitochondrial capacity.

Perilipin 2 Improves Insulin Sensitivity in Skeletal Muscle Despite Elevated Intramuscular Lipid Levels

Type 2 diabetes is characterized by excessive lipid storage in skeletal muscle. Excessive intramyocellular lipid (IMCL) storage exceeds intracellular needs and induces lipotoxic events, ultimately contributing to the development of insulin resistance. Lipid droplet (LD)–coating proteins may control proper lipid storage in skeletal muscle. Perilipin 2 (PLIN2/adipose differentiation–related protein [ADRP]) is one of the most abundantly expressed LD-coating proteins in skeletal muscle. Here we examined the role of PLIN2 in myocellular lipid handling and insulin sensitivity by investigating the effects of in vitro PLIN2 knockdown and in vitro and in vivo overexpression. PLIN2 knockdown decreased LD formation and triacylglycerol (TAG) storage, marginally increased fatty-acid (FA) oxidation, and increased incorporation of palmitate into diacylglycerols and phospholipids. PLIN2 overexpression in vitro increased intramyocellular TAG storage paralleled with improved insulin sensitivity. In vivo muscle-specific PLIN2 overexpression resulted in increased LD accumulation and blunted the high-fat diet–induced increase in protein content of the subunits of the oxidative phosphorylation (OXPHOS) chain. Diacylglycerol levels were unchanged, whereas ceramide levels were increased. Despite the increased IMCL accumulation, PLIN2 overexpression improved skeletal muscle insulin sensitivity. We conclude that PLIN2 is essential for lipid storage in skeletal muscle by enhancing the partitioning of excess FAs toward TAG storage in LDs, thereby blunting lipotoxicity-associated insulin resistance.

ATP8B1 is essential for maintaining normal hearing

ATP8B1 deficiency is caused by autosomal recessive mutations in ATP8B1, which encodes the putative phospatidylserine flippase ATP8B1 (formerly called FIC1). ATP8B1 deficiency is primarily characterized by cholestasis, but extrahepatic symptoms are also found. Because patients sometimes report reduced hearing capability, we investigated the role of ATP8B1 in auditory function. Here we show that ATP8B1/Atp8b1 deficiency, both in patients and in Atp8b1G308V/G308V mutant mice, causes hearing loss, associated with progressive degeneration of cochlear hair cells. Atp8b1 is specifically localized in the stereocilia of these hair cells. This indicates that the mechanosensory function and integrity of the cochlear hair cells is critically dependent on ATP8B1 activity, possibly through maintaining lipid asymmetry in the cellular membranes of stereocilia.

Augmenting muscle diacylglycerol and triacylglycerol content by blocking fatty acid oxidation does not impede insulin sensitivity

A low fat oxidative capacity has been linked to muscle diacylglycerol (DAG) accumulation and insulin resistance. Alternatively, a low fat oxidation rate may stimulate glucose oxidation, thereby enhancing glucose disposal. Here, we investigated whether an ethyl-2-[6-(4-chlorophenoxy)hexyl]-oxirane-2-carboxylate (etomoxir)-induced inhibition of fat oxidation leads to muscle fat storage and insulin resistance. An intervention in healthy male subjects was combined with studies in human primary myotubes. Furthermore, muscle DAG and triacylglycerol (TAG), mitochondrial function, and insulin signaling were examined in etomoxir-treated C57bl6 mice. In humans, etomoxir administration increased glucose oxidation at the expense of fat oxidation. This effect was accompanied by an increased abundance of GLUT4 at the sarcolemma and a lowering of plasma glucose levels, indicative of improved glucose homeostasis. In mice, etomoxir injections resulted in accumulation of muscle TAG and DAG, yet improved insulin-stimulated GLUT4 translocation. Also in human myotubes, insulin signaling was improved by etomoxir, in the presence of increased intramyocellular lipid accumulation. These insulin-sensitizing effects in mice and human myotubes were accompanied by increased phosphorylation of AMP-activated protein kinase (AMPK). Our results show that a reduction in fat oxidation leading to accumulation of muscle DAG does not necessarily lead to insulin resistance, as the reduction in fat oxidation may activate AMPK.

Paradoxical Increase in TAG and DAG Content Parallel the Insulin Sensitizing Effect of Unilateral DGAT1 Overexpression in Rat Skeletal Muscle

Background The involvement of muscle triacylglycerol (TAG) storage in the onset of insulin resistance is questioned and the attention has shifted towards inhibition of insulin signalling by the lipid intermediate diacylglycerol (DAG). The enzyme 1,2-acylCoA:diacylglyceroltransferase-1 (DGAT1) esterifies a fatty acyl-CoA on DAG to form TAG. Therefore, the aim of the present study was to investigate if unilateral overexpression of DGAT1 in adult rat Tibialis anterior (TA) muscle will increase conversion of the lipid intermediate DAG into TAG, thereby improving muscle insulin sensitivity. Methodology/Principal Findings The DGAT1 gene construct was injected in the left TA muscle of male rats on chow or high-fat (45% kcal) diet for three weeks, followed by application of one 800 V/cm and four 80 V/cm pulses, using the contralateral leg as sham-electroporated control. Seven days after electroporation, muscle specific insulin sensitivity was assessed with a hyperinsulinemic euglycemic clamp using 2-deoxy-[3H]glucose. Here, we provide evidence that unilateral overexpression of DGAT1 in TA muscle of male rats is associated with an increased rather than decreased DAG content. Strikingly, this increase in DAG content was accompanied by improved muscle insulin sensitivity. Interestingly, markers of muscle lipolysis and mitochondrial function were also increased in DGAT1 overexpressing muscle. Conclusions/Significance We conclude that unilateral DGAT1 overexpression can rescue insulin sensitivity, possibly by increasing DAG and TAG turnover in skeletal muscle. In case of a proper balance between the supply and oxidation of fatty acids in skeletal muscle, the lipid intermediate DAG may not exert harmful effects on insulin signalling.

Uncoupled respiration, ROS production, acute lipotoxicity and oxidative damage in isolated skeletal muscle mitochondria from UCP3-ablated mice

The function of uncoupling protein 3 (UCP3) is still not established. Mitochondrial uncoupling, control of ROS production, protection against lipotoxicity and protection against oxidative stress are functions classically discussed. To establish a role for UCP3 in these functions, we have here used UCP3 (-/-) mice, backcrossed for 10 generations on a C57Bl/6 background. In isolated skeletal muscle mitochondria, we examined uncoupled respiration, both unstimulated and in the presence of fatty acids. We did not observe any difference between mitochondria from wildtype and UCP3 (-/-) mice. We measured H(2)O(2) production rate and respiration rate under reactive oxygen species-generating conditions (succinate without rotenone) but found no effect of UCP3. We tested two models of acute lipotoxicity-fatty acid-induced oxidative inhibition and fatty acid-induced swelling-but did not observe any protective effect of UCP3. We examined oxidative stress by quantifying 4-hydroxynonenal protein adducts and protein carbonyls in the mitochondria-but did not observe any protective effect of UCP3. We conclude that under the experimental conditions tested here, we find no evidence for the function of UCP3 being basal or induced uncoupling, regulation of ROS production, protection against acute lipotoxicity or protection against oxidative damage.

High-fat diets rich in medium- versus long-chain fatty acids induce distinct patterns of tissue specific insulin resistance

Excess dietary long-chain fatty acid (LCFA) intake results in ectopic lipid accumulation and insulin resistance. Since medium-chain fatty acids (MCFA) are preferentially oxidized over LCFA, we hypothesized that diets rich in MCFA result in a lower ectopic lipid accumulation and insulin resistance compared to diets rich in LCFA. Feeding mice high-fat (HF) (45% kcal fat) diets for 8 weeks rich in triacylglycerols composed of MCFA (HFMCT) or LCFA (HFLCT) revealed a lower body weight gain in the HFMCT-fed mice. Indirect calorimetry revealed higher fat oxidation on HFMCT compared to HFLCT (0.011.0±0.0007 vs. 0.0096±0.0015 kcal/g body weight per hour, P<.05). In line with this, neutral lipid immunohistochemistry revealed significantly lower lipid storage in skeletal muscle (0.05±0.08 vs. 0.30±0.23 area%, P <.05) and in liver (0.9±0.4 vs. 6.4±0.8 area%, P<.05) after HFMCT vs. HFLCT, while ectopic fat storage in low fat (LF) was very low. Hyperinsulinemic euglycemic clamps revealed that the HFMCT and HFLCT resulted in severe whole body insulin resistance (glucose infusion rate: 53.1±6.8, 50.8±15.3 vs. 124.6±25.4 μmol min(-1) kg(-1), P<.001 in HFMCT, HFLCT and LF-fed mice, respectively). However, under hyperinsulinemic conditions, HFMCT revealed a lower endogenous glucose output (22.6±8.0 vs. 34.7±8.5 μmol min(-1) kg(-1), P<.05) and a lower peripheral glucose disappearance (75.7±7.8 vs. 93.4±12.4 μmol min(-1) kg(-1), P<.03) compared to HFLCT-fed mice. In conclusion, both HF diets induced whole body insulin resistance compared to LF. However, the HFMCT gained less weight, had less ectopic lipid accumulation, while peripheral insulin resistance was more pronounced compared to HFLCT. This suggests that HF-diets rich in medium- versus long-chain triacylglycerols induce insulin resistance via distinct mechanisms.

The Effects of Long- or Medium-Chain Fat Diets on Glucose Tolerance and Myocellular Content of Lipid Intermediates in Rats

Accumulation of triacylglycerols (TAGs) and acylcarnitines in skeletal muscle upon high‐fat (HF) feeding is the resultant of fatty acid uptake and oxidation and is associated with insulin resistance. As medium‐chain fatty acids (MCFAs) are preferentially β‐oxidized over long‐chain fatty acids, we examined the effects of medium‐chain TAGs (MCTs) and long‐chain TAGs (LCTs) on muscle lipid storage and whole‐body glucose tolerance. Rats fed a low‐fat (LF), HFLCT, or an isocaloric HFMCT diet displayed a similar body weight gain over 8 weeks of treatment. Only HFLCT increased myocellular TAG (42.3 ± 4.9, 71.9 ± 6.7, and 48.5 ± 6.5 µmol/g for LF, HFLCT, and HFMCT, respectively, P < 0.05) and long‐chain acylcarnitine content (P < 0.05). Neither HF diet increased myocellular diacylglycerol (DAG) content. Intraperitoneal (IP) glucose tolerance tests (1.5 g/kg) revealed a significantly decreased glucose tolerance in the HFMCT compared to the HFLCT‐fed rats (802 ± 40, 772 ± 18, and 886 ± 18 area under the curve for LF, HFLCT, and HFMCT, respectively, P < 0.05). Finally, no differences in myocellular insulin signaling after bolus insulin injection (10 U/kg) were observed between LF, HFLCT, or HFMCT‐fed rats. These results show that accumulation of TAGs and acylcarnitines in skeletal muscle in the absence of body weight gain do not impede myocellular insulin signaling or whole‐body glucose intolerance.

Significance of uncoupling protein 3 in mitochondrial function upon mid- and long-term dietary high-fat exposure

Uncoupling protein 3 (UCP3) may reduce mitochondrial ROS production, and thereby protect against mitochondrial dysfunction in skeletal muscle. UCP3 has been suggested to specifically fulfill this role under high‐fat conditions. Here we show that UCP3 knockout mice indeed have elevated mitochondrial ROS production after short‐term (8 weeks) high‐fat feeding. After 26 weeks of high‐fat feeding, UCP3 knockout mice exhibited reduced mitochondrial function as measured ex vivo in isolated mitochondria. In conclusion, these data suggest that UCP3 may have a role in the protection of mitochondria against lipid‐induced mitochondrial dysfunction, but only after long‐term exposure to high‐fat.

Palmitate-induced skeletal muscle insulin resistance does not require NF-κB activation.

Palmitate activates the NF-κB pathway, and induces accumulation of lipid metabolites and insulin resistance in skeletal muscle cells. Little information is available whether and how these processes are causally related. Therefore, the objectives were to investigate whether intra-cellular lipid metabolites are involved in FA-induced NF-κB activation and/or insulin resistance in skeletal muscle and to investigate whether FA-induced insulin resistance and NF-κB activation are causally related. Inhibiting DGAT or CPT-1 by using, respectively, amidepsine or etomoxir increased DAG accumulation and sensitized myotubes to palmitate-induced insulin resistance. While co-incubation of palmitate with etomoxir increased NF-κB transactivation, co-incubation with amidepsine did not, indicating that DAG accumulation is associated with insulin resistance but not with NF-κB activation. Furthermore, pharmacological or genetic inhibition of the NF-κB pathway could not prevent palmitate-induced insulin resistance. In conclusion, we have demonstrated that activation of the NF-κB pathway is not required for palmitate-induced insulin resistance in skeletal muscle cells.