Sean A. Newsom

Energy deficit after exercise augments lipid mobilization but does not contribute to the exercise-induced increase in insulin sensitivity

The content of meals consumed after exercise can impact metabolic responses for hours and even days after the exercise session. The purpose of this study was to compare the effect of low dietary carbohydrate (CHO) vs. low energy intake in meals after exercise on insulin sensitivity and lipid metabolism the next day. Nine healthy men participated in four randomized trials. During the control trial (CON) subjects remained sedentary. During the other three trials, subjects exercised [65% peak oxygen consumption (Vo(2 peak)); cycle ergometer and treadmill exercise] until they expended approximately 800 kcal. Dietary intake during CON and one exercise trial (BAL) was designed to provide sufficient energy and carbohydrate to maintain nutrient balance. In contrast, the diets after the other two exercise trials were low in either CHO (LOW-CHO) or energy (LOW-EN). The morning after exercise we obtained a muscle biopsy, assessed insulin sensitivity (S(i); intravenous glucose tolerance test) and measured lipid kinetics (isotope tracers). Although subjects were in energy balance during both LOW-CHO and CON, the lower muscle glycogen concentration during LOW-CHO vs. CON (402 +/- 29 vs. 540 +/- 33 mmol/kg dry wt, P < 0.01) coincided with a significant increase in S(i) [5.2 +/- 0.7 vs. 3.8 +/- 0.7 (mU/l)(-1) x min(-1); P < 0.05]. Conversely, despite ingesting several hundred fewer kilocalories after exercise during LOW-EN compared with BAL, this energy deficit did not affect S(i) the next day [4.9 +/- 0.9, and 5.0 +/- 0.8 (mU/l)(-1) x min(-1)]. Maintaining an energy deficit after exercise had the most potent effect on lipid metabolism, as measured by a higher plasma triacylglycerol concentration, and increased plasma fatty acid mobilization and oxidation compared with when in nutrient balance. Carbohydrate deficit after exercise, but not energy deficit, contributed to the insulin-sensitizing effects of acute aerobic exercise, whereas maintaining an energy deficit after exercise augmented lipid mobilization.

Skeletal Muscle MnSOD, Mitochondrial Complex II, and SIRT3 Enzyme Activities Are Decreased in Maternal Obesity During Human Pregnancy and Gestational Diabetes Mellitus

CONTEXT Insulin resistance and systemic oxidative stress are prominent features of pregnancies complicated by maternal obesity or gestational diabetes mellitus (GDM). The role of skeletal muscle oxidative stress or mitochondrial capacity in obese pregnant women or obese women with GDM is unknown. OBJECTIVE We investigated whether obese pregnant women, compared with normal weight (NW) pregnant women, demonstrate decreased skeletal muscle mitochondrial enzyme activity and elevated markers of oxidative stress, and if these differences are more severe in obese women diagnosed with GDM. DESIGN We measured mitochondrial enzyme activity and markers of oxidative stress in skeletal muscle tissue from NW pregnant women (n = 10), obese pregnant women with normal glucose tolerance (NGT; n = 10), and obese pregnant women with GDM (n = 8), undergoing cesarean delivery (∼37 wk gestation). RESULTS Electron transport complex-II and manganese superoxide dismutase (MnSOD) enzyme activities were decreased in obese-NGT and obese-GDM, compared with NW women. The glutathione redox ratio (GSH:GSSG) was decreased in obese-NGT and obese-GDM, indicative of increased oxidative stress. Mitochondrial sirtuin (SIRT)3 mRNA content and enzyme activity were lower in skeletal muscle of obese-NGT and obese-GDM women. Importantly, acetylation of MnSOD, a SIRT3 target, was increased in obese-NGT and obese-GDM vs NW women and was inversely correlated with SIRT3 activity (r = -0.603), suggesting a mechanism for reduced MnSOD activity. CONCLUSIONS These data show that obese pregnant women demonstrate decreased skeletal muscle mitochondrial respiratory chain enzyme activity and decreased mitochondrial antioxidant defense. Furthermore, reduced skeletal muscle SIRT3 activity may play a role in the increased oxidative stress associated with pregnancies complicated by obesity.

A Single Session of Low-Intensity Exercise Is Sufficient to Enhance Insulin Sensitivity Into the Next Day in Obese Adults

OBJECTIVE The purpose of this study was to determine the effect of a relatively modest session of exercise on insulin sensitivity and fatty acid uptake the next day in obese adults. RESEARCH DESIGN AND METHODS Eleven sedentary obese adults (male/female: 3/8; BMI 37 ± 1 kg/m2; peak oxygen uptake [VO2peak] 20 ± 1 mL/kg/min) completed three experimental trials. On two of these occasions, subjects exercised to expend 350 kcal in the afternoon. These two exercise trials were identical except for the exercise intensity (50% VO2peak [EX50] and 65% VO2peak [EX65]) and the duration of exercise necessary to expend 350 kcal (EX50 = ∼70 min; EX65 = ∼55 min). Subjects also completed a control trial (CON), without exercise. The next morning, we measured insulin sensitivity (hyperinsulinemic-euglycemic clamp) and whole-body fatty acid uptake (palmitate rate of disappearance from plasma [Rd]). RESULTS Exercise increased insulin sensitivity the next day, but whereas the 35% improvement after EX50 compared with CON was statistically significant (P = 0.01), the 20% improvement after EX65 was not (P = 0.17). Despite nearly identical values between CON and EX65 (P = 0.88), systemic fatty acid uptake was lower after EX50 compared with EX65 (P = 0.02), but not quite significant compared with CON (P = 0.07). Importantly, the change in fatty acid uptake after exercise compared with CON was negatively correlated with the change in insulin sensitivity for all trials (r = −0.60, P = 0.003). CONCLUSIONS A relatively modest single session of exercise in obese adults improved insulin sensitivity the next day, and a reduction in systemic fatty acid uptake in the several hours after exercise may be important for this effect.

Early Life Exposure to Maternal Insulin Resistance Has Persistent Effects on Hepatic NAFLD in Juvenile Nonhuman Primates

The origins of nonalcoholic fatty liver disease (NAFLD) may lie in early intrauterine exposures. Here we examined the maternal response to chronic maternal high-fat (HF) diet and the impact of postweaning healthy diet on mechanisms for NAFLD development in juvenile nonhuman primate (NHP) offspring at 1 year of age. Pregnant females on HF diet were segregated as insulin resistant (IR; HF+IR) or insulin sensitive (IS; HF+IS) compared with control (CON)-fed mothers. HF+IR mothers have increased body mass, higher triglycerides, and increased placental cytokines. At weaning, offspring were placed on a CON or HF diet. Only offspring from HF+IR mothers had increased liver triglycerides and upregulated pathways for hepatic de novo lipid synthesis and inflammation that was irreversible upon switching to a healthy diet. These juvenile livers also showed a combination of classical and alternatively activated hepatic macrophages and natural killer T cells, in the absence of obesity or insulin resistance. Our findings suggest that maternal insulin resistance, including elevated triglycerides, insulin, and weight gain, initiates dysregulation of the juvenile hepatic immune system and development of de novo lipogenic pathways that persist in vitro and may be an irreversible “first hit” in the pathogenesis of NAFLD in NHP.

High fatty acid availability after exercise alters the regulation of muscle lipid metabolism

We previously reported that a single exercise session protects against fatty acid (FA)-induced insulin resistance, perhaps in part through augmented intramyocellular triacylglycerol (IMTG) synthesis. The aim of this study was to examine the effect of elevated FA availability after exercise on factors regulating IMTG metabolism. After exercise (90 minutes, 65% peak oxygen uptake), 7 healthy women (body mass index, 23 ± 1 kg/m(2)) were infused overnight (16 hours) with either a lipid and heparin solution (LIPID, 0.11 g fat per kilogram per hour) or saline (SALINE). We measured resting FA oxidation (indirect calorimetry) and obtained a skeletal muscle biopsy sample the next morning. The 4-fold increase in overnight plasma FA concentration during LIPID increased IMTG by approximately 30% during LIPID vs SALINE. This was accompanied by an approximately 25% greater membrane-associated abundance of the FA transporter FAT/CD36 (P < .01) and an approximately 8% increase in the activity of the IMTG synthesis enzyme glycerol-3-phosphate acyltransferase (GPAT, P < .01). In contrast, resting FA oxidation was not affected. We also found no difference in the protein abundance of GPAT1 and diacylglycerol acyltransferase-1, diacylglycerol acyltransferase activity, or the abundance of the lipid droplet coat proteins (perilipins 2, 3, 4, and 5) between treatments. Our findings suggest that augmented capacity for FA flux into muscle (ie, via membrane-associated FAT/CD36), perhaps together with a slight yet significant increase in activity of a key IMTG synthesis enzyme (GPAT), may enhance IMTG storage when FA availability is high after exercise. The importance of the absence of a change in perilipin protein abundance despite increased muscle lipid storage remains to be determined.

Skeletal muscle phosphatidylcholine and phosphatidylethanolamine are related to insulin sensitivity and respond to acute exercise in humans

Several recent reports indicate that the balance of skeletal muscle phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is a key determinant of muscle contractile function and metabolism. The purpose of this study was to determine relationships between skeletal muscle PC, PE and insulin sensitivity, and whether PC and PE are dynamically regulated in response to acute exercise in humans. Insulin sensitivity was measured via intravenous glucose tolerance in sedentary obese adults (OB; n = 14), individuals with type 2 diabetes (T2D; n = 15), and endurance-trained athletes (ATH; n = 15). Vastus lateralis muscle biopsies were obtained at rest, immediately after 90 min of cycle ergometry at 50% maximal oxygen consumption (V̇o2 max), and 2-h postexercise (recovery). Skeletal muscle PC and PE were measured via infusion-based mass spectrometry/mass spectrometry analysis. ATH had greater levels of muscle PC and PE compared with OB and T2D (P < 0.05), with total PC and PE positively relating to insulin sensitivity (both P < 0.05). Skeletal muscle PC:PE ratio was elevated in T2D compared with OB and ATH (P < 0.05), tended to be elevated in OB vs. ATH (P = 0.07), and was inversely related to insulin sensitivity among the entire cohort (r = -0.43, P = 0.01). Muscle PC and PE were altered by exercise, particularly after 2 h of recovery, in a highly group-specific manner. However, muscle PC:PE ratio remained unchanged in all groups. In summary, total muscle PC and PE are positively related to insulin sensitivity while PC:PE ratio is inversely related to insulin sensitivity in humans. A single session of exercise significantly alters skeletal muscle PC and PE levels, but not PC:PE ratio.

Intracellular localization of diacylglycerols and sphingolipids influences insulin sensitivity and mitochondrial function in human skeletal muscle

BACKGROUND Accumulation of diacylglycerol (DAG) and sphingolipids is thought to promote skeletal muscle insulin resistance by altering cellular signaling specific to their location. However,the subcellular localization of bioactive lipids in human skeletal muscle is largely unknown. METHODS We evaluated subcellular localization of skeletal muscle DAGs and sphingolipids in lean individuals (n = 15), endurance-trained athletes (n = 16), and obese men and women with (n = 12) and without type 2 diabetes (n = 15). Muscle biopsies were fractionated into sarcolemmal, cytosolic, mitochondrial/ER, and nuclear compartments. Lipids were measured using liquid chromatography tandem mass spectrometry, and insulin sensitivity was measured using hyperinsulinemic-euglycemic clamp. RESULTS Sarcolemmal 1,2-DAGs were not significantly related to insulin sensitivity. Sarcolemmal ceramides were inversely related to insulin sensitivity, with a significant relationship found for the C18:0 species. Sarcolemmal sphingomyelins were also inversely related to insulin sensitivity, with the strongest relationships found for the C18:1, C18:0, and C18:2 species. In the mitochondrial/ER and nuclear fractions, 1,2-DAGs were positively related to, while ceramides were inversely related to, insulin sensitivity. Cytosolic lipids as well as 1,3-DAG, dihydroceramides, and glucosylceramides in any compartment were not related to insulin sensitivity. All sphingolipids but only specific DAGs administered to isolated mitochondria decreased mitochondrial state 3 respiration. CONCLUSION These data reveal previously unknown differences in subcellular localization of skeletal muscle DAGs and sphingolipids that relate to whole-body insulin sensitivity and mitochondrial function in humans. These data suggest that whole-cell concentrations of lipids obscure meaningful differences in compartmentalization and suggest that subcellular localization of lipids should be considered when developing therapeutic interventions to treat insulin resistance. FUNDING National Institutes of Health General Clinical Research Center (RR-00036), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01DK089170), NIDDK (T32 DK07658), and Colorado Nutrition Obesity Research Center (P30DK048520).

Long-term rates of mitochondrial protein synthesis are increased in mouse skeletal muscle with high fat feeding regardless of insulin sensitizing treatment

Skeletal muscle mitochondrial protein synthesis is regulated in part by insulin. The development of insulin resistance with diet-induced obesity may therefore contribute to impairments to protein synthesis and decreased mitochondrial respiration. Yet the impact of diet-induced obesity and insulin resistance on mitochondrial energetics is controversial, with reports varying from decreases to increases in mitochondrial respiration. We investigated the impact of changes in insulin sensitivity on long-term rates of mitochondrial protein synthesis as a mechanism for changes to mitochondrial respiration in skeletal muscle. Insulin resistance was induced in C57BL/6J mice using 4 wk of a high-fat compared with a low-fat diet. For 8 additional weeks, diets were enriched with pioglitazone to restore insulin sensitivity compared with nonenriched control low-fat or high-fat diets. Skeletal muscle mitochondrial protein synthesis was measured using deuterium oxide labeling during weeks 10-12 High-resolution respirometry was performed using palmitoyl-l-carnitine, glutamate+malate, and glutamate+malate+succinate as substrates for mitochondria isolated from quadriceps. Mitochondrial protein synthesis and palmitoyl- l-carnitine oxidation were increased in mice consuming a high-fat diet, regardless of differences in insulin sensitivity with pioglitazone treatment. There was no effect of diet or pioglitazone treatment on ADP-stimulated respiration or H2O2 emission using glutamate+malate or glutamate+malate+succinate. The results demonstrate no impairments to mitochondrial protein synthesis or respiration following induction of insulin resistance. Instead, mitochondrial protein synthesis was increased with a high-fat diet and may contribute to remodeling of the mitochondria to increase lipid oxidation capacity. Mitochondrial adaptations with a high-fat diet appear driven by nutrient availability, not intrinsic defects that contribute to insulin resistance.

Lipid mixtures containing a very high proportion of saturated fatty acids only modestly impair insulin signaling in cultured muscle cells.

In vitro examinations of the effect of saturated fatty acids on skeletal muscle insulin action often use only one or two different fatty acid species, which does not resemble the human plasma fatty acid profile. We compared graded concentrations (0.1-0.8mM) of 3 different lipid mixtures: 1) a physiologic fatty acid mixture (NORM; 40% saturated fatty acids), 2) a physiologic mixture high in saturated fatty acids (HSFA; 60% saturated fatty acids), and 3) 100% palmitate (PALM) on insulin signaling and fatty acid partitioning into triacylglycerol (TAG) and diacylglycerol (DAG) in cultured muscle cells. As expected, PALM readily impaired insulin-stimulated pAktThr308/Akt and markedly increased intracellular DAG content. In contrast, the fatty acid mixtures only modestly impaired insulin-stimulated pAktThr308M/Akt, and we found no differences between NORM and HSFA. Importantly, NORM and HSFA did not increase DAG content, but instead dose-dependently increased TAG accumulation. Therefore, the robust impairment in insulin signaling found with palmitate exposure was attenuated with physiologic mixtures of fatty acids, even with a very high proportion of saturated fatty acids. This may be explained in part by selective partitioning of fatty acids into neutral lipid (i.e., TAG) when muscle cells were exposed to physiologic lipid mixtures.

Relatively low endogenous fatty acid mobilization and uptake helps preserve insulin sensitivity in obese women.

Background:Although obesity is commonly linked with metabolic disease risk, some obese adults do not develop metabolic abnormalities, such as insulin resistance.Objectives:The primary aim of this study was to determine whether alterations in fatty acid mobilization and uptake underlie differences in insulin sensitivity (Si) among a seemingly homogeneous cohort of obese women.Methods:Insulin sensitivity (frequently sampled intravenous glucose tolerance test), basal fatty acid rate of disappearance from plasma (Rd), resting whole-body fat oxidation, intramyocellular triacylglycerol (IMTG) concentration and markers of skeletal muscle inflammation were measured in 21 obese women. Participants were divided into tertiles based on their Si. The subset of participants with the lowest Si (LOW-Si; Si⩽2.1 (mU/l)−1 min−1; n=7) was compared with the subset of participants with the highest Si, who exhibited relatively normal insulin sensitivity (NORM-Si; Si⩾3.4 (mU/l)−1 min−1; n=8).Results:Despite nearly identical physical characteristics in LOW-Si vs NORM-Si (body mass index: 34±2 vs 34±1 kg m−2; %body fat: 48±1 vs 47±1%; waist circumference: 104±2 vs 104±2 cm; VO2 max: 2.2±0.2 vs 2.3±0.1 l min−1), fatty acid Rd was nearly 30% lower in NORM (P=0.02). Importantly, the greater rate of fatty acid uptake in LOW-Si vs NORM-Si did not translate to higher rate of fat oxidation (3.5±0.2 vs 3.7±0.2 μmol kg−1 min−1) or to a measureable difference in IMTG content (68.3±12.7 vs 63.7±6.7 μmol g−1 dry weight). In conjunction with the lower fatty acid Rd in NORM-Si vs LOW-Si, activation of inflammatory pathways known to impair insulin action in skeletal muscle was also lower (lower phosphorylated c-jun N-terminal kinase (JNK) and higher inhibitor of κB (IκB-α) abundance). In contrast, LOW-Si and NORM-Si exhibited no differences in plasma markers of inflammation (TNFα, IL-6 (interleukin-6), MCP-1).Conclusion:These findings suggest that obese women who maintain a relatively low rate of endogenous fatty acid uptake may be somewhat ‘protected’ against the development of insulin resistance potentially by less activation of inflammatory pathways within skeletal muscle.