Agnieszka Mikłosz

Fatty acid transporters involved in the palmitate and oleate induced insulin resistance in primary rat hepatocytes.

To determine the presence and possible involvement of FAT/CD36, FABPpm and FATP‐2, transporters in (i) fatty acids movement across plasma membrane and (ii) an induction of insulin resistance by palmitic (PA) and oleic (OA) fatty acids in primary hepatocytes.

Effects of Inhibition of Serine Palmitoyltransferase (SPT) and Sphingosine Kinase 1 (SphK1) on Palmitate Induced Insulin Resistance in L6 Myotubes

Background The objective of this study was to examine the effects of short (2 h) and prolonged (18 h) inhibition of serine palmitoyltransferase (SPT) and sphingosine kinase 1 (SphK1) on palmitate (PA) induced insulin resistance in L6 myotubes. Methods L6 myotubes were treated simultaneously with either PA and myriocin (SPT inhibitor) or PA and Ski II (SphK1inhibitor) for different time periods (2 h and 18 h). Insulin stimulated glucose uptake was measured using radioactive isotope. Expression of insulin signaling proteins was determined using Western blot analyses. Intracellular sphingolipids content [sphinganine (SFA), ceramide (CER), sphingosine (SFO), sphingosine-1-phosphate (S1P)] were estimated by HPLC. Results Our results revealed that both short and prolonged time of inhibition of SPT by myriocin was sufficient to prevent ceramide accumulation and simultaneously reverse palmitate induced inhibition of insulin-stimulated glucose transport. In contrast, prolonged inhibition of SphK1 intensified the effect of PA on insulin-stimulated glucose uptake and attenuated further the activity of insulin signaling proteins (pGSK3β/GSK3β ratio) in L6 myotubes. These effects were related to the accumulation of sphingosine in palmitate treated myotubes. Conclusion Myriocin is more effective in restoration of palmitate induced insulin resistance in L6 myocytes, despite of the time of SPT inhibition, comparing to SKII (a specific SphK1 inhibitor). Observed changes in insulin signaling proteins were related to the content of specific sphingolipids, namely to the reduction of ceramide. Interestingly, inactivation of SphK1 augmented the effect of PA induced insulin resistance in L6 myotubes, which was associated with further inhibition of insulin stimulated PKB and GSK3β phosphorylation, glucose uptake and the accumulation of sphingosine.

Inhibition of Ceramide De Novo Synthesis Ameliorates Diet Induced Skeletal Muscles Insulin Resistance

Nowadays wrong nutritional habits and lack of physical activity give a rich soil for the development of insulin resistance and obesity. Many researches indicate lipids, especially the one from the sphingolipids class, as the group of molecules heavily implicated in the progress of insulin resistance in skeletal muscle. Recently, scientists have focused their scrutiny on myriocin, a potent chemical compound that inhibits ceramide (i.e., central hub of sphingolipids signaling pathway) de novo synthesis. In the present research we evaluated the effects of myriocin application on type 2 diabetes mellitus in three different types of skeletal muscles: (1) slow-oxidative (red gastrocnemius), (2) oxidative-glycolytic (soleus), and (3) glycolytic (white gastrocnemius). For these reasons the animals were randomly divided into four groups: “control” (C), “myriocin” (M), “high fat diet” (HFD), “high fat diet” (HFD), and “high fat diet + myriocin” (HFD + M). Our in vivo study demonstrated that ceramide synthesis inhibition reduces intramuscular ceramide, its precursor sphinganine, and its derivatives sphingosine and sphingosine-1-phosphate concentrations. Moreover, FFA and TG contents were also decreased after myriocin treatment. Thus, myriocin presents potential therapeutic perspectives with respect to the treatment of insulin resistance and its serious consequences in obese patients.

Modest Decrease in Pgc1α Results in TAG Accumulation but not in Insulin Resistance in L6 Myotubes

Background/Aims: PGC-1α is an important cellular protein (coactivator) regulating myocyte mitochondria number and function, and therefore whole cellular energy status. The aim of this work was to investigate the effects of modest, temporary PGC-1α knock-down on L6 myotubes insulin resistance in a light of cellular lipid metabolism. Methods: Gas liquid chromatography was applied for assessing FAs content and composition. For the expression of mitochondrial enzymes, as well as FA and glucose transporters, Western Blot technique was adopted. Additionally, radiolabelled glucose and palmitic acid uptake was performed to estimate the nutrients cellular influx. Results: Modest (-24%) PGC-1α protein ablation resulted in decreased mitochondrial activity in general (reduced Cyt C content) and FAs oxidation in particular (diminished β-HAD expression) without increased FAs cellular influx. The aforementioned intervention led to significantly increased TAG cellular level, but not DAG nor CER. Consequently, no changes in cellular insulin responsiveness were noticed. Conclusions: Modest (-24%) PGC-1α protein depletion results in lipid accumulation, without causing insulin resistance. Importantly, it seems that this TAG loading is a result of decreased mitochondrial oxidative capacity and/or possibly increased lipid biosynthesis but not fatty acid cellular influx.

The Role of PGC-1α in the Development of Insulin Resistance in Skeletal Muscle - Revisited

Currently, obesity is a predominant medical condition and an important risk factor for the development of several diseases, including type 2 diabetes mellitus. Importantly, most research has indicated lipid-induced insulin resistance in skeletal muscles is a key link between the aforementioned pathological conditions. PGC-1α is a prominent regulator of myocellular energy metabolism orchestrating gene transcription programming in response to numerous environmental stimuli. Moreover, it is widely acknowledged that mitochondrial metabolism (primary metabolic target of PGC-1α) disturbances are widely acknowledged contributors to type 2 diabetes development. Therefore, it seems surprising that the exact physiological contribution of PGC-1α in the development of insulin resistance in skeletal muscle remains poorly understood. This review aims to reconcile these allegedly different findings by looking for a common denominator in the role(s) of PGC-1α in respect to lipid-induced insulin resistance in skeletal muscle. Our scrutiny of the literature indicates that interventions at the level of PGC-1α may exert beneficial effects on myocytes in respect to lipid-induced insulin resistance. The latter takes place as a result of a positive net energy balance (fatty acids oxidation surpassing their accumulation rate). Moreover, the aforementioned effects may not necessarily be limited to physically active states. They seem to occur, however, only within a physiologically observed range in muscle cells (approximately 1-fold changes in PGC-1α protein expression).

Challenging of AS160/TBC1D4 Alters Intracellular Lipid Milieu in L6 Myotubes Incubated with Palmitate.

The Akt substrate of 160 kDa (AS160) is a key regulator of GLUT4 translocation from intracellular depots to the plasma membrane in myocytes. Likely, AS160 also controls LCFAs transport, which requires relocation of fatty acid transporters. The aim of the present study was to determine the impact of AS160 knockdown on lipid milieu in L6 myotubes incubated with palmitate (PA). Therefore, we compared two different settings, namely: 1) AS160 knockdown prior to palmitate incubation (pre‐PA‐silencing, AS160−/PA); 2) palmitate incubation with subsequent AS160 knockdown (post‐PA‐silencing, PA/AS160−). The efficiency of AS160 silencing was checked at mRNA and protein levels. The expression and localization of FA transporters were determined using Western Blot and immunofluorescence analyses. Intracellular lipid content (FFA, DAG, TAG, and PL) and FA composition were estimated by GLC, whereas basal palmitate uptake was analyzed by means of scintigraphy. Both groups with silenced AS160 were characterized by a greater expression of FA transporters (FAT/CD36, FATP‐1, 4) which had contributed to an increased FA cellular influx. Accordingly, we observed that post‐PA‐silencing of AS160 resulted in a marked decrement in DAG, TAG, and PL contents, but increased FFA content (PA/AS160− vs. PA). The opposite effect was observed in the group with pre‐PA‐silencing of AS160 in which AS160 knockdown did not affect the lipid pools (AS160−/PA vs. PA). Our results indicate that post‐PA‐silencing of AS160 has a capacity to decrease the lipotoxic effect(s) of PA by decreasing the content of lipids (DAG and PL) that promote insulin resistance in myotubes. J. Cell. Physiol. 232: 2373–2386, 2017.

Fiber specific changes in sphingolipid metabolism in skeletal muscles of hyperthyroid rats.

Thyroid hormones (T3, T4) are well known modulators of different cellular signals including the sphingomyelin pathway. However, studies regarding downstream effects of T3 on sphingolipid metabolism in skeletal muscle are scarce. In the present work we sought to investigate the effects of hyperthyroidism on the activity of the key enzymes of ceramide metabolism as well as the content of fundamental sphingolipids. Based on fiber/metabolic differences, we chose three different skeletal muscles, with diverse fiber compositions: soleus (slow-twitch oxidative), red (fast-twitch oxidative-glycolytic) and white (fast-twitch glycolytic) section of gastrocnemius. We demonstrated that T3 induced accumulation of sphinganine, ceramide, sphingosine, as well as sphingomyelin, mostly in soleus and in red, but not white section of gastrocnemius. Concomitantly, the activity of serine palmitoyltransferase and acid/neutral ceramidase was increased in more oxidative muscles. In conclusion, hyperthyroidism induced fiber specific changes in the content of sphingolipids that were relatively more related to de novo synthesis of ceramide rather than to its generation via hydrolysis of sphingomyelin.

Akumulacja lipidów (triacylo-, diacylogliceroli i ceramidów) wewnątrz hepatocytów, a rozwój insulinooporności wątrobowej

Insulin resistance (IR) is commonly defined as a lack of insulin effects on target tissues, due to impaired post-receptor signaling pathways. Generally, liver IR is manifested by uncontrolled glucose release to the blood stream (hyperglycemia). However, metabolic consequences of hepatic insulin resistance are more profound, involving also lipid imbalances. Accumulation of intracellular lipids such as diacylglycerols (DAG) and ceramides (CER) was found to interfere directly with the insulin signaling cascade, inducing hepatic IR. Molecular targets of elevated DAG and/ or CER levels include activation of protein kinase C (PKC) and/or protein phosphatase that dephosphorylates Akt/PKB. In either case as a result insulin resistance develops, enhancing hyperglycemia and subsequent hyperinsulinemia, which in turn aggravate liver lipogenesis and fatty acid accumulation.

Sphingolipids metabolism in the salivary glands of rats with obesity and streptozotocin induced diabetes

Diabetes is considered a major public health problem affecting millions of individuals worldwide. Remarkably, scientific reports regarding salivary glands sphingolipid metabolism in diabetes are virtually non‐existent. This is odd given the well‐established link between the both in other tissues (e.g., skeletal muscles, liver) and the key role of these glands in oral health preservation. The aim of this paper is to examine sphingolipids metabolism in the salivary glands in (pre)diabetes (evoked by high fat diet feeding or streptozotocin). Wistar rats were allocated into three groups: control, HFD‐, or STZ‐diabetes. The content of major sphingolipid classes in the parotid (PSG) and submandibular (SMSG) glands was assessed via chromatography. Additionally, Western blot analyses were employed for the evaluation of key sphingolipid signaling pathway enzyme levels. No changes in ceramide content in the PSG were found, whereas an increase in ceramide concentration for SMSG of the STZ group was observed. This was accompanied by an elevation in SPT1 level. Probably also sphingomyelin hydrolysis was increased in the SMSG of the STZ‐diabetic rats, since we observed a significant drop in the amount of SM. PSG and SMSG respond differently to (pre)diabetes, with clearer pattern presented by the later gland. An activation of sphingomyelin signaling pathway was observed in the course of STZ‐diabetes, that is, metabolic condition with rapid onset/progression. Whereas, chronic HFD lead to an inhibition of sphingomyelin signaling pathway in the salivary glands (manifested in an inhibition of ceramide de novo synthesis and accumulation of S1P).

The Effects of AS160 Modulation on Fatty Acid Transporters Expression and Lipid Profile in L6 Myotubes

Background/Aims: AS160 is a key intracellular regulator of energy utilization in cells. It was shown to regulate GLUT4 translocation from intracellular depots to the plasma membrane, with subsequent changes in facilitated glucose uptake into the skeletal muscles. Similarly, also free fatty acids (FFAs) transmembrane transport seems to be largely protein-mediated. Therefore, the objective of this study was to examine the effects of moderate AS160 depletion (-82% mRNA, -25% of protein content) on the expression of fatty acid transporters and subsequent changes in lipid profile in L6 myotubes. Results: Surprisingly, moderate down regulation of AS160 expression was followed by increased AS160 phosphorylation (∼40%). These resulted in a greater expression of fatty acid transporters, namely FABPpm and FAT/CD36, with subsequently increased FAs cellular influx. No changes in the expression of FATP1 and 4 were noticed. Accordingly, we have observed a reduction in total TAG content. This was mainly caused by a significant changes in TAG fatty acids composition favouring a decrease in the amount of palmitic and stearic fatty acid moieties. In contrast, our experimental intervention led to distinctively increased total content of DAG and PL, but concomitantly decreased the content of all measured sphingolipids, e.g. SFA, SA1P, CER, SFO and S1P, in the AS160 knockdown group. Conclusions: Modulation of AS160 level and activity led to significant increase in the concentration of DAG and PL, which was associated with changes in FAs composition and expression of fatty acid transporters. Interestingly, the intervention also simultaneously decreased the content of sphingolipids.