Modulation of adipokine production, glucose uptake and lactate release in human adipocytes by small changes in oxygen tension

Adipose tissue becomes hypoxic in obesity, and cell culture studies have demonstrated that hypoxia leads to major changes in adipocyte function. Studies on the response of adipocytes to low O₂ tension have employed marked hypoxia (1% O₂). Here, we have examined the effects of modest hypoxia, utilising differing concentrations of O₂ (1–21%), on adipokine production and glucose uptake by human adipocytes. Incubation with 10% O₂ (24 h) increased expression of the leptin, vascular endothelial growth factor (VEGF) and Angptl4 genes, while leptin expression was elevated even at 15% O₂ (compared to ‘normoxia’—21% O₂). Overall, there was a concentration-dependent increase in the expression of these genes as O₂ fell, with the highest mRNA level evident at 1% O₂. Parallel changes were observed in the secretion of leptin, VEGF and IL-6 into the medium, an increased release being evident at 10% O₂ (15% O₂ for leptin). Adiponectin gene expression was reduced at 15% O₂ and below, while adiponectin release was significantly reduced at 5% O₂. Both 2-deoxy-d-glucose uptake and lactate release showed progressive increases as O₂ concentration fell, being significantly raised at 10% and 5% O₂, respectively. The alterations in substrate transport were accompanied by parallel changes in transporter gene expression, GLUT1 and MCT1 mRNA level increasing from 15% and 10% O₂, respectively. These results indicate that marked responses to reduced O₂ concentration are exhibited by human adipocytes at O₂ levels well above those associated with hypoxia and employed in cell culture studies. Adipocytes are sensitive to small changes in O₂ tension.     

Expression of monocarboxylate transporter (MCT) 1 and MCT4 in overloaded mice plantaris muscle

A number of studies have shown that changes in muscle contractile activity regulate the expression of monocarboxylate transporters (MCTs) in the skeletal muscle. The aim of this study was to investigate the effect of functional overload on MCT1 and MCT4 protein expression. Plantaris muscles were functionally overloaded for 15 days by ablation of the synergistic muscles. MCT1 and MCT4 mRNA abundance increased by 160–161% (p < 0.01) and 265–325% (p < 0.05), respectively, after 1–3 days of functional overload. MCT1 and MCT4 protein expression increased by 92 and 61%, respectively, after 12 days of functional overload (p < 0.05). AMP-activated protein kinase (AMPK) phosphorylation status [phospho-AMPK (Thr172)/total AMPK] was significantly elevated after 3–9 days of functional overload. Plasma testosterone concentration was elevated after 12 days of functional overload, while blood lactate concentration was not altered. Thus, the current study demonstrated that heavy mechanical loading induces increase in MCT1 and MCT4 protein expression in the muscles with increase in AMPK phosphorylation status and plasma testosterone concentration.     

Cell-specific localization of monocarboxylate transporters, MCT1 and MCT2, in the adult mouse brain revealed by double immunohistochemical labeling and confocal microscopy

Recent evidence suggests that lactate could be a preferential energy substrate transferred from astrocytes to neurons. This would imply the presence of specific transporters for lactate on both cell types. We have investigated the immunohistochemical localization of two monocarboxylate transporters, MCT1 and MCT2, in the adult mouse brain. Using specific antibodies raised against MCT1 and MCT2, we found strong immunoreactivity for each transporter in glia limitans, ependymocytes and several microvessel-like elements. In addition, small processes distributed throughout the cerebral parenchyma were immunolabeled for monocarboxylate transporters. Double immunofluorescent labeling and confocal microscopy examination of these small processes revealed no co-localization between glial fibrillary acidic protein and monocarboxylate transporters, although many glial fibrillary acidic protein-positive processes were often in close apposition to elements labeled for monocarboxylate transporters. In contrast, several elements expressing the S100beta protein, another astrocytic marker found to be located in distinct parts of the same cell when compared with glial fibrillary acidic protein, were also strongly immunoreactive for MCT1, suggesting expression of this transporter by astrocytes. In contrast, MCT2 was expressed in a small subset of microtubule-associated protein-2-positive elements, indicating a neuronal localization.In conclusion, these observations are consistent with the possibility that lactate, produced and released by astrocytes (via MCT1), could be taken up (via MCT2) and used by neurons as an energy substrate.     

Monocarboxylate Transporter 1 (MCT1) is an independent prognostic biomarker in endometrial cancer

Background

Endometrial cancer (EC) is a major health concern due to its rising incidence. Whilst early stage disease is generally cured by surgery, advanced EC has a poor prognosis with limited treatment options. Altered energy metabolism is a hallmark of malignancy. Cancer cells drive tumour growth through aerobic glycolysis and must export lactate to maintain intracellular pH. The aim of this study was to evaluate the expression of the lactate/proton monocarboxylate transporters MCT1 and MCT4 and their chaperone CD147 in EC, with the ultimate aim of directing future drug development.

Methods

MCT1, MCT4 and CD147 expression was examined using immunohistochemical analysis in 90 endometrial tumours and correlated with clinico-pathological characteristics and survival outcomes.

Results

MCT1 and MCT4 expression was observed in the cytoplasm, the plasma membrane or both locations. CD147 was detected in the plasma membrane and associated with MCT1 (p =?0.003) but not with MCT4 (p =?0.207) expression. High MCT1 expression was associated with reduced overall survival (p =?0.029) and remained statistically significant after adjustment for survival covariates (p =?0.017).

Conclusion

Our data suggest that MCT1 expression is an important marker of poor prognosis in EC. MCT1 inhibition may have potential as a treatment for advanced or recurrent EC.

     

Effects of strength training on muscle lactate release and MCT1 and MCT4 content in healthy and type 2 diabetic humans

Lactate is released from skeletal muscle in proportion to glucose uptake rates, and it leaves the cells via simple diffusion and two monocarboxylate transporter proteins, MCT1 and MCT4. In reponse to endurance training MCT1 – and possibly MCT4 – content in muscle increases. The MCTs have not previously been measured in patients with type 2 diabetes (Type 2), and the response to strength training is unknown. Ten Type 2 and seven healthy men (Control) strength-trained one leg (T) 3 times a week for 6 weeks while the other leg remained untrained (UT). Each session lasted no more than 30 min. After strength training, muscle biopsies were obtained and an isoglycaemic, hyperinsulinaemic clamp, combined with arterial and femoral venous catheterization of both legs, was carried out. During hyperinsulinaemia lactate release was always increased in T versus UT legs. MCT1 was lower ( P <0.05) and MCT4 similar in Type 2 versus Control. With training, MCT1 content always increased, while MCT4 only increased in Control. Conclusions: MCT1 content in skeletal muscle in Type 2 is lower compared with healthy men. Strength training increases MCT1 content in healthy men and in Type 2, thus normalizing the content in Type 2.     

Microarray analysis identifies matrix metalloproteinases (MMPs) as key genes whose expression is up-regulated in human adipocytes by macrophage-conditioned medium

White adipose tissue exhibits inflammation as tissue mass expands in obesity, involving macrophage infiltration and a direct inflammatory response by adipocytes. DNA microarrays and conditioned medium have been used to examine the effects of macrophages on global gene expression in human adipocytes. SGBS adipocytes, differentiated in culture, were treated with macrophage-conditioned medium (U937 cells) for 4 or 24 h; control cells received unconditioned medium. Agilent arrays comprising 44,000 probes were used to analyse gene expression. Microarray analysis identified 1,088 genes differentially expressed in response to the conditioned medium at both 4 and 24 h (754 up-regulated, 334 down-regulated at 24 h); these included genes associated with inflammation and macrophage infiltration. A cluster of matrix metalloproteinase genes were highly up-regulated at both time-points, including MMP1, MMP3, MMP9, MMP10, MMP12 and MMP19. At 4 and 24 h, MMP1 was the most highly up-regulated gene (>2,400-fold increase in mRNA at 24 h). ELISA measurements indicated that substantial quantities of MMP1 and MMP3 were released from adipocytes incubated with conditioned medium, with little release by control adipocytes. Treatment with TNFα induced substantial increases in MMP1 (>100-fold) and MMP3 (27-fold) mRNA level and MMP1 and MMP3 release in adipocytes, suggesting that this cytokine could contribute to the stimulation of MMP expression by macrophages. In conclusion, macrophage-secreted factors induce a major inflammatory response in human adipocytes, with expression of MMP family members being strongly up-regulated. The induction of MMP1 and other MMPs suggests that macrophages stimulate tissue remodelling during adipose tissue expansion in obesity.     

Effect of AMPK activation on monocarboxylate transporter (MCT)1 and MCT4 in denervated muscle

It is now evident that exercise training leads to increases in monocarboxylate transporter (MCT)1 and MCT4, but little is known about the mechanisms of coupling muscle contraction with these changes. The aim of this study was to investigate the effect of 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) induced activation of AMP-activated protein kinase (AMPK) on MCT1, MCT4, and GLUT4 in denervated muscle. Protein levels of MCT4 and GLUT4 after 10 days of denervation were significantly decreased in mice gastrocnemius muscle, while MCT1 protein levels were not altered. AICAR treatment for 10 days significantly increased MCT4, and GLUT4 protein levels in innervated muscle as shown in previous studies. We found that the MCT1 protein level was also increased in AICAR treated innervated muscle. AICAR treatment prevented the decline in MCT4 and GLUT4 protein levels in denervated muscle. Thus, the current study suggests that MCT1 and MCT4 protein expression in muscles, as well as GLUT4, may be regulated by AMPK-mediated signal pathways, and AMPK activation can prevent denervation-induced decline in MCT4 protein.     

Effects of hypoxia on cerebral and muscle haemodynamics during knee extensions in healthy subjects

A hypoxic model was used to investigate changes in localized cerebral and muscle haemodynamics during knee extension (KE) in healthy individuals. Thirty-one young healthy volunteers performed one set of KE until failure under hypoxia (14 % O₂) or normoxia (21 % O₂) at 50, 75 or 100 % of 1 repetition maximum, in random order, on three occasions. Prefrontal cerebral and vastus lateralis muscle oxygenation and blood volume (Cox, Mox, Cbv and Mbv, respectively) were recorded simultaneously by near-infrared spectroscopy. Hypoxia induced significant declines in Cox [?0.017 ± 0.016 optical density (OD) units] and Mox (?0.014 ± 0.026 OD units) and increases in Cbv (0.017 ± 0.027 OD units) and Mbv (0.016 ± 0.023 OD units) at rest. Hypoxia significantly reduced total work (TW) performed during KE at each exercise intensity. Cox, Cbv, Mox, and Mbv changes during KE did not differ between normoxia and hypoxia. Correlations between TW done and Cox changes under normoxia (r = 0.04, p = 0.182) and hypoxia (r = 0.05, p = 0.122) were not significant. However, TW was significantly correlated with Mox under both normoxia (R ² = 0.24, p = 0.000) and hypoxia (R ² = 0.15, p = 0.004). Since changes in Cox and Mox reflect alterations in the balance between oxygen delivery and extraction in these tissues, which, in the brain, is an index of neuronal activation, we conclude that: (1) limitation of KE performance was mediated peripherally under both normoxia and hypoxia, with no additional effect of hypoxia, and (2) because of the low common variance with Mox additional intramuscular factors likely play a role in limiting KE performance.     

Effect of hypoxia on arterial and venous blood levels of oxygen,carbon dioxide,hydrogen ions and lactate during incremental forearm exercise

Summary The purpose of the present study was to investigate whether, in humans, hypoxia results in an elevated lactate production from exercising skeletal muscle. Under conditions of both hypoxia [inspired oxygen fraction (F_IO₂): 11.10%] and normoxia (F_IO₂: 20.94%), incremental exercise of a forearm was performed. The exercise intensity was increased every minute by 1.6 kg·m·min^–1 until exhaustion. During the incremental exercise the partial pressure of oxygen (PO₂) and carbon dioxide (PCO₂), oxygen saturation (SO₂), pH and lactate concentration [HLa] of five subjects, were measured repeatedly in blood from the brachial artery and deep veins from muscles in the forearm of both the active and inactive sides. The hypoxia (arterial SO₂ approximately 70%) resulted in (1) the difference in [HLa] in venous blood from active muscle (values during exercise — resting value) often being more than twice that for normoxia, (2) a significantly greater difference in venous-arterial (v-a) [HLa] for the exercising muscle compared to normoxia, and (3) a difference in v-a [HLa] for non-exercising muscle that was slightly negative during normoxia and more so with hypoxia. These studies suggest that lower O₂ availability to the exercising muscle results in increased lactate production.     

Current aspects of lactate exchange: lactate/H+ transport in human skeletal muscle

Skeletal muscle is capable of producing and releasing large amounts of lactate and at the same time taking up lactate and using it as a respiratory fuel. The release and uptake of lactate both involve transmembrane transport, which is mediated mainly by a membrane protein called the monocarboxylate transporter (MCT). MCTs mediate membrane transport with an obligatory 1:1 coupling between lactate and H⁺ fluxes, and is therefore of great importance for pH regulation, especially during intense muscle activity. The total lactate and H⁺ transport capacity is higher in membranes from oxidative fibers than in membranes from more glycolytic fibers. There are two isoforms of MCT present in skeletal muscle, MCT1 and MCT4. In human muscle samples, there is a positive correlation between the proportion of type I fibers and MCT1 density. In contrast, the MCT4 density in human muscle is independent of fiber type and displays a large interindividual variation. Although the two isoforms have identical transport kinetics (K _m), they may have different roles in muscle. MCT1 and MCT4 respond differently to a high-intensity training session, which suggests that these two isoforms are regulated differently. Electronic Publication     

Effect of low oxygen inhalation on changes in blood pH, lactate, and ammonia due to exercise

The present study examined the effect of hypoxia-induced respiratory alkalosis on exercise-induced metabolic acidosis and increases in plasma lactate and ammonia levels. Six male subjects underwent exercise of increasing intensity until exhaustion: (1) in normoxia (20.9% O₂) (=MAX), (2) in hypoxia (12% O₂) (=HP) in which hypoxic condition had been maintained from 60 min before to 30 min after exercise, and (3) the same intensity of exercise as HP in normoxia (=SUB). Arterialized blood was drawn from a superficial vein. Post-exercise blood pH was significantly higher in HP than in MAX (P<0.05), although plasma lactate was at the same level. For hypoxia as compared to normoxia, regression analysis confirmed a parallel shift of plasma lactate to higher pH levels indicating the effect of respiratory alkalosis (P<0.01). After exercise plasma levels of ammonia were lower in HP than in MAX (P<0.05). Regression analysis between ammonia and pH revealed nearly identical changes in hypoxia and normoxia at low pH. From these results, we conclude that: (1) hypoxia-induced respiratory alkalosis attenuated exhaustive exercise-induced metabolic acidosis, (2) plasma lactate concentration was determined by the relative exercise intensity, (3) the maximum plasma ammonia concentration under exhaustive exercise was reduced at hypoxia because of respiratory alkalosis.     

Effect of acute hypoxia on muscle blood flow, VO2p, and [HHb] kinetics during leg extension exercise in older men

The adjustment of pulmonary oxygen uptake (VO_2p), heart rate (HR), limb blood flow (LBF), and muscle deoxygenation [HHb] was examined during the transition to moderate-intensity, knee-extension exercise in six older adults (70 ± 4 years) under two conditions: normoxia (FIO₂ = 20.9 %) and hypoxia (FIO₂ = 15 %). The subjects performed repeated step transitions from an active baseline (3 W) to an absolute work rate (21 W) in both conditions. Phase 2 VO_2p, HR, LBF, and [HHb] data were fit with an exponential model. Under hypoxic conditions, no change was observed in HR kinetics, on the other hand, LBF kinetics was faster (normoxia 34 ± 3 s; hypoxia 28 ± 2), whereas the overall [HHb] adjustment ( $ \tau^{\prime } = {\text{TD}} + \tau $ ) was slower (normoxia 28 ± 2; hypoxia 33 ± 4 s). Phase 2 VO_2p kinetics were unchanged (p < 0.05). The faster LBF kinetics and slower [HHb] kinetics reflect an improved matching between O₂ delivery and O₂ utilization at the microvascular level, preventing the phase 2 VO_2p kinetics from become slower in hypoxia. Moreover, the absolute blood flow values were higher in hypoxia (1.17 ± 0.2 L min^?1) compared to normoxia (0.96 ± 0.2 L min^?1) during the steady-state exercise at 21 W. These findings support the idea that, for older adults exercising at a low work rate, an increase of limb blood flow offsets the drop in arterial oxygen content (CaO₂) caused by breathing an hypoxic mixture.     

Hypoxia Increases Serum Amyloid A3 (SAA3) in Differentiated 3T3-L1 Adipocytes

Hypoxia has been implicated as a possible cause of adipose tissue inflammation. Furthermore, the acute phase protein serum amyloid A (SAA) has been associated with the modulation of the adipogenic process, and it is well-known that obese individuals have increased levels of SAA. The effect of hypoxia in the expression and production of SAA was examined in murine 3T3-L1 adipocytes. Hypoxia leads to a substantial increase in SAA3 mRNA and protein level, apparently in a time-dependent manner (threefold in 48 h), in fully differentiated 3T3-L1, followed by reestablishment of gene expression to basal levels after 24 h of reoxygenation. Hypoxia-induced SAA may be one of the key molecules to the development of the inflammatory response in adipose tissue.