Arterio-venous balance studies of skeletal muscle fatty acid metabolism: What can we believe?

The arterio-venous balance (A-V balance/difference) technique has been used by a number of groups, including ours, to study skeletal muscle fatty acid metabolism. Several lines of evidence indicate that, like glycogen, intramyocellular triglycerides (imcTG) are an energy source for local use. As such, the report that increased release of free fatty acids (FFA) via lipolysis from skeletal muscle, but not from adipose tissue, is responsible for the increased systemic lipolysis during IL-6 infusion in healthy humans is somewhat unexpected (26). It appears that given the complex anatomy of human limbs, as to be discussed in this review, it is virtually impossible to determine whether any fatty acids being released into the venous circulation of an arm or leg derive from the lipolysis of intermuscular fat residing between muscle groups, intramuscular fat residing within muscle groups (between epimysium and perimysium, or bundles), or the intramyocellular triglyceride droplets (imcTG). In many cases, it may even be difficult to be confident that there is no contribution of FFA from subcutaneous adipose tissue. This question is fundamentally important as one attempts to interpret the results of skeletal muscle fatty acid metabolism studies using the A-V balance technique. In this Perspectives article, we examine the reported results of fatty acid kinetics obtained using the techniques to evaluate the degree of and how to minimize contamination when attempting to sample skeletal muscle-specific fatty acids.

Hyperinsulinemia and skeletal muscle fatty acid trafficking.

We hypothesized that insulin alters plasma free fatty acid (FFA) trafficking into intramyocellular (im) long-chain acylcarnitines (imLCAC) and triglycerides (imTG). Overnight-fasted adults (n = 41) received intravenous infusions of [U-¹³C]palmitate (0400-0900 h) and [U-¹³C]oleate (0800-1400 h) to label imTG and imLCAC. A euglycemic-hyperinsulinemic (1.0 mU·kg fat-free mass⁻¹·min⁻¹) clamp (0800-1400 h) and two muscle biopsies (0900 h, 1400 h) were performed. The patterns of [U-¹³C]palmitate incorporation into imTG-palmitate and palmitoylcarnitine were similar to those we reported in overnight postabsorptive adults (saline control); the intramyocellular palmitoylcarnitine enrichment was not different from and correlated with imTG-palmitate enrichment for both the morning (r = 0.38, P = 0.02) and afternoon (r = 0.44, P = 0.006) biopsy samples. Plasma FFA concentrations, flux, and the incorporation of plasma oleate into imTG-oleate during hyperinsulinemia were ~1/10th of that observed in the previous saline control studies (P < 0.001). At the time of the second biopsy, the enrichment in oleoylcarnitine was <25% of that in imTG-oleate and was not correlated with imTG-oleate enrichment. The intramyocellular nonesterified fatty acid-palmitate-to-imTG-palmitate enrichment ratio was greater (P < 0.05) in women than men, suggesting that sex differences in intramyocellular palmitate trafficking may occur under hyperinsulinemic conditions. We conclude that plasma FFA trafficking into imTG during hyperinsulinemia is markedly suppressed, and these newly incorporated FFA fatty acids do not readily enter the LCAC preoxidative pools. Hyperinsulinemia does not seem to inhibit the entry of fatty acids from imTG pools that were labeled under fasting conditions, possibly reflecting the presence of two distinct imTG pools that are differentially regulated by insulin.

Relationship between plasma free fatty acid, intramyocellular triglycerides and long-chain acylcarnitines in resting humans.

We hypothesized that plasma non-esterified fatty acids (NEFA) are trafficked directly to intramyocellular long-chain acylcarnitines (imLCAC) rather than transiting intramyocellular triglycerides (imTG) on the way to resting muscle fatty acid oxidation. Overnight fasted adults (n = 61) received intravenous infusions of [U-(13)C]palmitate (0400-0830 h) and [U-(13)C]oleate (0800-1400 h) labelling plasma NEFA, imTG, imLCAC and im-non-esterified FA (imNEFA). Two muscle biopsies (0830 and 1400 h) were performed following 6 h, overlapping, sequential palmitate/oleate tracer infusions. Enrichment of plasma palmitate was approximately 15 times greater than enrichment of imTG, imNEFA-palmitate and im-palmitoyl-carnitine. Fatty acid enrichment in LCAC was correlated with imTG and imNEFA; there was a significant correlation between imTG concentrations and imLCAC concentrations in women (r = 0.51, P = 0.005), but not men (r = 0.30, P = 0.11). We estimated that approximately 11% of NEFA were stored in imTG. imTG NEFA storage was correlated only with NEFA concentrations (r = 0.52, P = 0.004) in women and with V(O(2),peak) (r = 0.45, P = 0.02) in men. At rest, plasma NEFA are trafficked largely to imTG before they enter LCAC oxidative pools; thus, imTG are an important, central pool that regulates the delivery of fatty acids to the intracellular environment. Factors relating to plasma NEFA storage into imTG differ in men and women.

Peroxisome proliferator-activated receptor gamma agonism modifies the effects of growth hormone on lipolysis and insulin sensitivity.

CONTEXT: Peroxisome proliferator-activated receptor gamma (PPAR-gamma) agonists such as thiazolidinediones (TZDs) improve insulin sensitivity in type 2 diabetes mellitus (T2DM) through effects on fat metabolism whereas GH stimulates lipolysis and induces insulin resistance. OBJECTIVE: To evaluate the impact of TZDs on fat metabolism and insulin sensitivity in subjects exposed to stable GH levels. DESIGN: A randomized, placebo-controlled, double-blind parallel-group study including 20 GH-deficient patients on continued GH replacement therapy. The patients were studied before and after 12 weeks. INTERVENTION: Patients received either pioglitazone 30 mg (N = 10) or placebo (N = 10) once daily for 12 weeks. RESULTS: Adiponectin levels almost doubled during pioglitazone treatment (P = 0.0001). Pioglitazone significantly decreased basal free fatty acid (FFA) levels (P = 0.02) and lipid oxidation (P = 0.02). Basal glucose oxidation rate (P = 0.004) and insulin sensitivity (P = 0.03) improved in the patients who received pioglitazone treatment. The change in insulin-stimulated adiponectin level after pioglitazone treatment was positively correlated to the change in insulin-stimulated total glucose disposal (R = 0.69, P = 0.04). CONCLUSION: The impact of GH on lipolysis and insulin sensitivity can be modified by administration of TZDs.

Intramyocellular lipids: maker vs. marker of insulin resistance.

Intramyocellular triglyceride (imcTG) content in skeletal muscle is abnormally high in lipid oversupply models in obesity, type 2 diabetes (T2D) and other metabolically diseased conditions. The imcTG abnormality was also found to be significantly correlated with muscle insulin resistance (MIR). As skeletal muscle is the main site for insulin-mediated glucose utilization, the research on this topic has been active since. However, to date the pathways responsible for the imcTG excess and the mechanisms underlying the imcTG-MIR correlation have not been identified. A current view is focused on a backward mechanism that fatty acid oxidation by muscle is impaired causing imcTG to accumulate and, therefore, an enlarged imcTG pool is merely a marker of MIR. However, based on kinetic studies, it is more likely that imcTG is a source of MIR. On one hand, an enlarged and fast turning over imcTG pool interferes with insulin signaling by producing excess amounts of signaling molecules that activate PKC pathways. On the other hand, it may promote mitochondrial beta-oxidation that suppresses glucose metabolism via substrate competition. Therefore, it is hypothesized that imcTG is a source of MIR.

Clearance kinetics of the VGF-derived neuropeptide TLQP-21.

TLQP-21 is a multifunctional neuropeptide and a promising new medicinal target for cardiometabolic and neurological diseases. However, to date its clearance kinetics and plasma stability have not been studied. The presence of four arginine residues led us to hypothesize that its half-life is relatively short. Conversely, its biological activities led us to hypothesize that the peptide is still taken up by adipose tissues effectively. [125I]TLQP-21 was i.v. administered in rats followed by chasing the plasma radioactivity and assessing tissue uptake. Plasma stability was measured using LC-MS. In vivo lipolysis was assessed by the palmitate rate of appearance. RESULTS: A small single i.v. dose of [125I]TLQP-21 had a terminal half-life of 110 min with a terminal clearance rate constant, kt, of 0.0063/min, and an initial half-life of 0.97 min with an initial clearance rate constant, ki, of 0.71/min. The total net uptake by adipose tissue accounts for 4.4% of the entire dose equivalent while the liver, pancreas and adrenal gland showed higher uptake. Uptake by the brain was negligible, suggesting that i.v.-injected peptide does not cross the blood-brain-barrier. TLQP-21 sustained isoproterenol-stimulated lipolysis in vivo. Finally, TLQP-21 was rapidly degraded producing several N-terminal and central sequence fragments after 10 and 60 min in plasma in vitro. This study investigated the clearance and stability of TLQP-21 peptide for the first time. While its pro-lipolytic effect supports and extends previous findings, its short half-life and sequential cleavage in the plasma suggest strategies for chemical modifications in order to enhance its stability and therapeutic efficacy.

Skeletal muscle mitochondrial functions, mitochondrial DNA copy numbers, and gene transcript profiles in type 2 diabetic and nondiabetic subjects at equal levels of low or high insulin and euglycemia.

We investigated whether previously reported muscle mitochondrial dysfunction and altered gene transcript levels in type 2 diabetes might be secondary to abnormal blood glucose and insulin levels rather than an intrinsic defect of type 2 diabetes. A total of 13 type 2 diabetic and 17 nondiabetic subjects were studied on two separate occasions while maintaining similar insulin and glucose levels in both groups by 7-h infusions of somatostatin, low- or high-dose insulin (0.25 and 1.5 mU/kg of fat-free mass per min, respectively), and glucose. Muscle mitochondrial DNA abundance was not different between type 2 diabetic and nondiabetic subjects at both insulin levels, but the majority of transcripts in muscle that are involved mitochondrial functions were expressed at lower levels in type 2 diabetes at low levels of insulin. However, several gene transcripts that are specifically involved in the electron transport chain were expressed at higher levels in type 2 diabetic patients. After the low-dose insulin infusion, which achieved postabsorptive insulin levels, the muscle mitochondrial ATP production rate (MAPR) was not different between type 2 diabetic and nondiabetic subjects. However, increasing insulin to postprandial levels increased the MAPR in nondiabetic subjects but not in type 2 diabetic patients. The lack of MAPR increment in response to high-dose insulin in type 2 diabetic patients occurred in association with reduced glucose disposal and expression of peroxisome proliferator-activated receptor-gamma coactivator 1alpha, citrate synthase, and cytochrome c oxidase I. In conclusion, the current data supports that muscle mitochondrial dysfunction in type 2 diabetes is not an intrinsic defect, but instead a functional defect related to impaired response to insulin.

Splanchnic lipolysis in human obesity.

Elevated FFA concentrations have been shown to reproduce some of the metabolic abnormalities of obesity. It has been hypothesized that visceral adipose tissue lipolysis releases excess FFAs into the portal vein, exposing the liver to higher FFA concentrations. We used isotope dilution/hepatic vein catheterization techniques to examine whether intra-abdominal fat contributes a greater portion of hepatic FFA delivery in visceral obesity. Obese women (n = 24) and men (n = 20) with a range of obesity phenotypes, taken together with healthy, lean women (n = 12) and men (n = 12), were studied. Systemic, splanchnic, and leg FFA kinetics were measured. The results showed that plasma FFA concentrations were approximately 20% greater in obese men and obese women. The contribution of splanchnic lipolysis to hepatic FFA delivery ranged from less than 10% to almost 50% and increased as a function of visceral fat in women (r = 0.49, P = 0.002) and in men (r = 0.52, P = 0.002); the slope of the relationship was greater in women than in men (P < 0.05). Leg and splanchnic tissues contributed a greater portion of systemic FFA release in obese men and women than in lean men and women. We conclude that the contribution of visceral adipose tissue lipolysis to hepatic FFA delivery increases with increasing visceral fat in humans and that this effect is greater in women than in men.

Energy expenditure, sex, and endogenous fuel availability in humans.

Adipose tissue lipolysis supplies circulating FFAs, which largely meet lipid fuel needs; however, excess FFAs, can contribute to the adverse health consequences of obesity. Because "normal" FFA release has not been well defined, average (mean of 4 days) basal FFA release and its potential regulation factors were measured in 50 lean and obese adults (25 women). Resting energy expenditure (REE), but not body composition, predicted most of the interindividual variation in FFA release. There was a significant, positive linear relationship between palmitate release and REE; however, women released approximately 40% more FFA than men relative to REE. Neither plasma palmitate concentrations nor respiratory quotient by indirect calorimetry differed between men and women. Glucose release rates were not different in men and women whether related to REE or fat free mass. These findings indicate that nonoxidative FFA clearance is greater in women than in men. This could be an advantage at times of increased fuel needs. We conclude that "normal" adipose tissue lipolysis is different in men and women and that the fuel export role of adipose tissue in obesity will need to be reassessed.

Intramyocellular lipid kinetics and insulin resistance.

More than fifteen years ago it was discovered that intramyocellular triglyceride (imcTG) content in skeletal muscle is abnormally high in conditions of lipid oversupply (e.g. high fat feeding) and, later, obesity, type 2 diabetes (T2D) and other metabolic conditions. This imcTG excess is robustly associated with muscle insulin resistance (MIR). However, to date the pathways responsible for the imcTG excess and the mechanisms underlying the imcTG-MIR correlation remain unclear. A current hypothesis is based on a backward mechanism that impaired fatty acid oxidation by skeletal muscle causes imcTG to accumulate. As such, imcTG excess is considered a marker but not a player in MIR. However, recent results from kinetic studies indicated that imcTG pool in high fat-induced obesity (HFO) model is kinetically dynamic. On one hand, imcTG synthesis is accelerated and contributes to imcTG accumulation. On the other, the turnover of imcTG is also accelerated. A hyperdynamic imcTG pool can impose dual adverse effects on glucose metabolism in skeletal muscle. It increases the release and thus the availability of fatty acids in myocytes that may promote fatty acid oxidation and suppress glucose utilization. Meanwhile, it releases abundant fatty acid products (e.g. diacylglycerol, ceramides) that impair insulin actions via signal transduction, thereby causing MIR. Thus, intramyocellular fatty acids and their products released from imcTG appear to function as a link to MIR. Accordingly, a forward mechanism is proposed that explains the imcTG-MIR correlation.

The neuropeptide TLQP-21 opposes obesity via C3aR1-mediated enhancement of adrenergic-induced lipolysis.

OBJECTIVES: Obesity is characterized by excessive fat mass and is associated with serious diseases such as type 2 diabetes. Targeting excess fat mass by sustained lipolysis has been a major challenge for anti-obesity therapies due to unwanted side effects. TLQP-21, a neuropeptide encoded by the pro-peptide VGF (non-acronymic), that binds the complement 3a receptor 1 (C3aR1) on the adipocyte membrane, is emerging as a novel modulator of adipocyte functions and a potential target for obesity-associated diseases. The molecular mechanism is still largely uncharacterized. METHODS: We used a combination of pharmacological and genetic gain and loss of function approaches. 3T3-L1 and mature murine adipocytes were used for in vitro experiments. Chronic in vivo experiments were conducted on diet-induced obese wild type, ß1, ß2, ß3-adrenergic receptor (AR) deficient and C3aR1 knockout mice. Acute in vivo lipolysis experiments were conducted on Sprague Dawley rats. RESULTS: We demonstrated that TLQP-21 does not possess lipolytic properties per se. Rather, it enhances ß-AR activation-induced lipolysis by a mechanism requiring Ca2+ mobilization and ERK activation of Hormone Sensitive Lipase (HSL). TLQP-21 acutely potentiated isoproterenol-induced lipolysis in vivo. Finally, chronic peripheral TLQP-21 treatment decreases body weight and fat mass in diet induced obese mice by a mechanism involving ß-adrenergic and C3a receptor activation without associated adverse metabolic effects. CONCLUSIONS: In conclusion, our data identify an alternative pathway modulating lipolysis that could be targeted to diminish fat mass in obesity without the side effects typically observed when using potent pro-lipolytic molecules.

Intrasample variability of intramyocellular triacylglycerol.

Intrasample variability of intramyocellular triacylglycerol (imcTG) in the skeletal muscle of rats has been examined. Aliquoting after homogenization of muscle samples reduced imcTG variability considerably compared with aliquoting before homogenization. The results suggested that skeletal muscle samples be homogenized before aliquoting in order to reduce imcTG variability.

Evidence for increased and insulin-resistant lipolysis in skeletal muscle of high-fat-fed rats.

The metabolic and isotopic profiles of glycerol in skeletal muscle were examined using awake, fasted lean and high-fat-induced obese rats, and hyperinsulinemic-euglycemic clamp was performed to assess the effect of insulin. During the clamp, Intralipid (no heparin; Fresnius Kabi Clayton, Clayton, NC), free fatty acids, glycerol, and glucose were coinfused to maintain their respective basal plasma levels in both groups. At steady-state, [U-(14)C]glycerol was infused intravenously for 120 minutes followed by muscle biopsy. The classical phenotypic characteristics of obesity, namely, reduced insulin-stimulated glucose uptake, a failure to suppress systemic lipolysis by insulin, and elevated plasma fatty acid concentration, were observed in the obese rats. Novel observations showed that in the basal state, the isotopic specific activity (S.A.) of glycerol (dpm/nmol) in gastrocnemius (0.03 v 0.12), soleus (0.05 v 0.12), and tibialis anterior (0.03 v 0.12) was significantly lower (all P <.003) in obese than in lean rats despite similar concentrations, indicating an active basal intramyocellular lipolysis. In addition, the lipolysis appeared resistant to insulin because the suppression of muscle glycerol during the clamp was 8%, 12%, and 8% in obese compared to 67%, 71%, and 63% in the lean control for gastrocnemius (P =.001), soleus (P =.007), and tibialis anterior (P =.004), respectively. The active intracellular lipolysis likely disturbs metabolic functions that may contribute to insulin resistance.

Substrate concentration and metabolism in left and right muscles of rats.

Skeletal muscle fiber heterogeneity among muscle groups is well known; however, laterality of muscle metabolism has not been addressed. In the present studies, metabolite concentrations in left and right gastrocnemius, tibialis anterior, quadriceps, and soleus muscles and their response to exogenous insulin have been compared in fasted awake rats. The results indicated that the concentrations of muscle free glycerol (P >.4), glycerol 3-phosphate (P >.1) nonesterified fatty acids (NEFA) (P >.6) and intramyocellular triglycerides (imcTG) (P >.08) are comparable between left and right of the same muscle, and are similar among mixed glycolytic-oxidative muscles. The concentration of free glycerol in soleus responded to exogenous insulin in a pattern distinct to that seen for the mixed muscles. The results support interchangeable use of left and right side of same muscles, and probably among different muscles of similar fiber type, but not muscles of different fiber types.

Determination of skeletal muscle triglyceride synthesis using a single muscle biopsy.

A novel stable isotopic technique for the determination of triglyceride synthesis in skeletal muscle by using a single muscle biopsy has been developed and evaluated in rats. In previous studies using (13)C-tracers, muscle triglyceride synthesis is usually determined using at least 2 biopsies, the first of which serves as the baseline sample for the measurement of natural (13)C abundance. In the present studies, the baseline biopsy has been eliminated by making the use of the isotopic information of a nontraced fatty acid in the muscle triglyceride pool. This is based on the fact that the source and, hence, the natural (13)C abundance of fatty acids in the same triglyceride pool is similar. To demonstrate and validate the method, a series of rat studies have been conducted to have established that (1) the natural (13)C abundance of 4 major fatty acids in the muscle triglyceride pool is similar; (2) there are no (13)C-label exchanges between fatty acids in the lipid pool; and (3) the incorporation of (13)C-palmitate into muscle triglycerides determined using this technique favorably compared with that determined by the traditional method. This approach makes stable isotope studies possible in which more than 1 muscle biopsy is difficult or impossible. Therefore, it has the potential to facilitate investigation of triglyceride metabolism in the skeletal muscle.

Dual tail catheters for infusion and sampling in rats as an efficient platform for metabolic experiments.

The authors present a technique for establishing a virtually stress-free dual-catheter system in rats, allowing researchers to conduct extended, complex experimental procedures involving the simultaneous infusion of multiple compounds and frequent collection of blood samples.

Reducing Liver Fat by Low Carbohydrate Caloric Restriction Targets Hepatic Glucose Production in Non-Diabetic Obese Adults with Non-Alcoholic Fatty Liver Disease.

UNLABELLED: Non-alcoholic fatty liver disease (NAFLD) impairs liver functions, the organ responsible for the regulation of endogenous glucose production and thus plays a key role in glycemic homeostasis. Therefore, interventions designed to normalize liver fat content are needed to improve glucose metabolism in patients affected by NAFLD such as obesity. OBJECTIVE: this investigation is designed to determine the effects of caloric restriction on hepatic and peripheral glucose metabolism in obese humans with NAFLD. METHODS: eight non-diabetic obese adults were restricted for daily energy intake (800 kcal) and low carbohydrate (<10%) for 8 weeks. Body compositions, liver fat and hepatic glucose production (HGP) and peripheral glucose disposal before and after the intervention were determined. RESULTS: the caloric restriction reduced liver fat content by 2/3 (p = 0.004). Abdominal subcutaneous and visceral fat, body weight, BMI, waist circumference and fasting plasma triglyceride and free fatty acid concentrations all significantly decreased (p < 0.05). The suppression of post-load HGP was improved by 22% (p = 0.002) whereas glucose disposal was not affected (p = 0.3). Fasting glucose remained unchanged and the changes in the 2-hour plasma glucose and insulin concentration were modest and statistically insignificant (p > 0.05). Liver fat is the only independent variable highly correlated to HGP after the removal of confounders. CONCLUSION: NAFLD impairs HGP but not peripheral glucose disposal; low carbohydrate caloric restriction effectively lowers liver fat which appears to directly correct the HGP impairment.

Locating the source of hyperglycemia: liver versus muscle.

BACKGROUND: Glucose homeostasis relies on insulin to suppress hepatic glucose production and to stimulate glucose uptake by peripheral tissues (primarily skeletal muscle) during and after a meal or glucose load. Glucose metabolism impairments in the liver and/or muscle attenuate these insulin actions, causing hyperglycemia. Thus, identifying the loci of the impairments can improve the understanding of hyperglycemia and enable organ-targeted interventions. METHODS: Studies were performed to identify such loci using modified oral glucose tolerance test (OGTT) techniques in individuals with type 2 diabetes (T2D) and overweight/obese individuals. RESULTS: Individuals with severe T2D were found to have significantly impaired glucose metabolism in both the liver and muscle. In contrast, impairments in glucose metabolism in individuals with non-severe T2D were predominantly localized in the liver or muscle, but not both. Similarly, milder impairments in overweight or obese individuals were clearly localized in either the liver or muscle, but not both. All these impairments are quantifiable. CONCLUSION: Impairments in glucose metabolism in the liver and muscle can be differentiated and quantified in a clinical setting.