Insulin deficiency and intranasal insulin alter brain mitochondrial function: a potential factor for dementia in diabetes.

Despite the strong association between diabetes and dementia, it remains to be fully elucidated how insulin deficiency adversely affects brain functions. We show that insulin deficiency in streptozotocin-induced diabetic mice decreased mitochondrial ATP production and/or citrate synthase and cytochrome oxidase activities in the cerebrum, hypothalamus, and hippocampus. Concomitant decrease in mitochondrial fusion proteins and increased fission proteins in these brain regions likely contributed to altered mitochondrial function. Although insulin deficiency did not cause any detectable increase in reactive oxygen species (ROS) emission, inhibition of monocarboxylate transporters increased ROS emission and further reduced ATP production, indicating the causative roles of elevated ketones and lactate in counteracting oxidative stress and as a fuel source for ATP production during insulin deficiency. Moreover, in healthy mice, intranasal insulin administration increased mitochondrial ATP production, demonstrating a direct regulatory role of insulin on brain mitochondrial function. Proteomics analysis of the cerebrum showed that although insulin deficiency led to oxidative post-translational modification of several proteins that cause tau phosphorylation and neurofibrillary degeneration, insulin administration enhanced neuronal development and neurotransmission pathways. Together these results render support for the critical role of insulin to maintain brain mitochondrial homeostasis and provide mechanistic insight into the potential therapeutic benefits of intranasal insulin.-Ruegsegger, G. N., Manjunatha, S., Summer, P., Gopala, S., Zabeilski, P., Dasari, S., Vanderboom, P. M., Lanza, I. R., Klaus, K. A., Nair, K. S. Insulin deficiency and intranasal insulin alter brain mitochondrial function: a potential factor for dementia in diabetes.

A novel triple-tracer approach to assess postprandial protein turnover.

Insulin and nutrients have profound effects on proteome homeostasis. Currently no reliable methods are available to measure postprandial protein turnover. A triple-tracer method was developed using phenylalanine stable isotope tracers to estimate appearance rates of ingested (Ra meal) and endogenous phenylalanine and the rate of phenylalanine disposal (Rd). This was compared with the "traditional" dual-tracer method, using one (1-CM)- and two (2-CM)-compartment models. For both methods, [13C6]phenylalanine was given orally, and [15N]phenylalanine was constantly infused; the triple-tracer method added [2H5]phenylalanine, infused at rates to mimic meal [13C6]phenylalanine appearance. Additionally, incorporation of meal-derived phenylalanine into specific proteins was measured after purification by two-dimensional electrophoresis. The triple-tracer approach reduced modeling errors, allowing improved reconstruction of Ra meal with a tracer-to-tracee ratio that was more constant and better estimated Rd. The 2-CM better described phenylalanine kinetics and Rd than 1-CM. Thus, the triple-tracer approach using 2-CM is superior for measuring non-steady-state postprandial protein turnover. This novel approach also allows measurement of postprandial synthesis rates of specific plasma proteins. We offer a valid non-steady-state model to measure postprandial protein turnover and synthesis of plasma proteins that can safely be applied in adults, children, and pregnant women.

FOXO3a regulates BNIP3 and modulates mitochondrial calcium, dynamics, and function in cardiac stress.

The forkhead box O3a (FOXO3a) transcription factor has been shown to regulate glucose metabolism, muscle atrophy, and cell death in postmitotic cells. Its role in regulation of mitochondrial and myocardial function is not well studied. Based on previous work, we hypothesized that FOXO3a, through BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3), modulates mitochondrial morphology and function in heart failure (HF). We modulated the FOXO3a-BNIP3 pathway in normal and phenylephrine (PE)-stressed adult cardiomyocytes (ACM) in vitro and developed a cardiotropic adeno-associated virus serotype 9 encoding dominant-negative FOXO3a (AAV9.dn-FX3a) for gene delivery in a rat model of HF with preserved ejection fraction (HFpEF). We found that FOXO3a upregulates BNIP3 expression in normal and PE-stressed ACM, with subsequent increases in mitochondrial Ca2+, leading to decreased mitochondrial membrane potential, mitochondrial fragmentation, and apoptosis. Whereas dn-FX3a attenuated the increase in BNIP3 expression and its consequences in PE-stressed ACM, AAV9.dn-FX3a delivery in an experimental model of HFpEF decreased BNIP3 expression, reversed adverse left ventricular remodeling, and improved left ventricular systolic and, particularly, diastolic function, with improvements in mitochondrial structure and function. Moreover, AAV9.dn-FX3a restored phospholamban phosphorylation at S16 and enhanced dynamin-related protein 1 phosphorylation at S637. Furthermore, FOXO3a upregulates maladaptive genes involved in mitochondrial apoptosis, autophagy, and cardiac atrophy. We conclude that FOXO3a activation in cardiac stress is maladaptive, in that it modulates Ca2+ cycling, Ca2+ homeostasis, and mitochondrial dynamics and function. Our results suggest an important role of FOXO3a in HF, making it an attractive potential therapeutic target.

Application of high-resolution mass spectrometry to measure low abundance isotope enrichment in individual muscle proteins.

Stable isotope-labeled amino acids have long been used to measure the fractional synthesis rate of proteins, although the mass spectrometry platforms used for such analyses have changed throughout the years. More recently, tandem mass spectrometers such as triple quadrupoles have been accepted as the standard platform for enrichment measurement due to their sensitivity and the enhanced specificity offered by multiple reaction monitoring (MRM) experiments. The limit in the utility of such platforms for enrichment analysis occurs when measuring very low levels of enrichment from small amounts of sample, particularly proteins isolated from two-dimensional gel electrophoresis (2D-GE), where interference from contaminant ions impacts the sensitivity of the measurement. We therefore applied a high-resolution orbitrap mass spectrometer to the analysis of [ring-(13)C6]-phenylalanine enrichment in individual muscle proteins isolated with 2D-GE. Comparison of samples analyzed on both platforms revealed that the high-resolution MS has significantly improved sensitivity relative to the triple quadrupole MS at very low-level enrichments due to its ability to resolve interferences in the m/z dimension. At higher enrichment levels, enrichment measurements from the orbitrap platform showed significant correlation (R (2) > 0.5) with those of the triple quadrupole platform. Together, these results indicate that high-resolution MS platforms such as the orbitrap are not only as capable of performing isotope enrichment measurements as the more commonly preferred triple quadrupole instruments, but offer unparalleled advantages in terms of mass accuracy and sensitivity in the presence of similar-mass contaminants.

Impact of insulin deprivation and treatment on sphingolipid distribution in different muscle subcellular compartments of streptozotocin-diabetic C57Bl/6 mice.

Insulin deprivation in type 1 diabetes (T1D) individuals increases lipolysis and plasma free fatty acids (FFA) concentration, which can stimulate synthesis of intramyocellular bioactive lipids such as ceramides (Cer) and long-chain fatty acid-CoAs (LCFa-CoAs). Ceramide was shown to decrease muscle insulin sensitivity, and at mitochondrial levels it stimulates reactive oxygen species production. Here, we show that insulin deprivation in streptozotocin diabetic C57BL/6 mice increases quadriceps muscle Cer content, which was correlated with a concomitant decrease in the body fat and increased plasma FFA, glycosylated hemoglobin level (%Hb A1c), and muscular LCFa-CoA content. The alternations were accompanied by an increase in protein expression in LCFa-CoA and Cer synthesis (FATP1/ACSVL5, CerS1, CerS5), a decrease in the expression of genes implicated in muscle insulin sensitivity (GLUT4, GYS1), and inhibition of insulin signaling cascade by Aktα and GYS3ß phosphorylation under acute insulin stimulation. Both the content and composition of sarcoplasmic fraction sphingolipids were most affected by insulin deprivation, whereas mitochondrial fraction sphingolipids remained stable. The observed effects of insulin deprivation were reversed, except for content and composition of LCFa-CoA, CerS protein expression, GYS1 gene expression, and phosphorylation status of Akt and GYS3ß when exogenous insulin was provided by subcutaneous insulin implants. Principal component analysis and Pearson's correlation analysis revealed close relationships between the features of the diabetic phenotype, the content of LCFa-CoAs and Cers containing C18-fatty acids in sarcoplasm, but not in mitochondria. Insulin replacement did not completely rescue the phenotype, especially regarding the content of LCFa-CoA, or proteins implicated in Cer synthesis and muscle insulin sensitivity. These persistent changes might contribute to muscle insulin resistance observed in T1D individuals.

Divergent Skeletal Muscle Metabolomic Signatures of Different Exercise Training Modes Independently Predict Cardiometabolic Risk Factors.

We investigated the link between enhancement of SI (by hyperinsulinemic-euglycemic clamp) and muscle metabolites after 12 weeks of aerobic (high-intensity interval training [HIIT]), resistance training (RT), or combined training (CT) exercise in 52 lean healthy individuals. Muscle RNA sequencing revealed a significant association between SI after both HIIT and RT and the branched-chain amino acid (BCAA) metabolic pathway. Concurrently with increased expression and activity of branched-chain ketoacid dehydrogenase enzyme, many muscle amino metabolites, including BCAAs, glutamate, phenylalanine, aspartate, asparagine, methionine, and γ-aminobutyric acid, increased with HIIT, supporting the substantial impact of HIIT on amino acid metabolism. Short-chain C3 and C5 acylcarnitines were reduced in muscle with all three training modes, but unlike RT, both HIIT and CT increased tricarboxylic acid metabolites and cardiolipins, supporting greater mitochondrial activity with aerobic training. Conversely, RT and CT increased more plasma membrane phospholipids than HIIT, suggesting a resistance exercise effect on cellular membrane protection against environmental damage. Sex and age contributed modestly to the exercise-induced changes in metabolites and their association with cardiometabolic parameters. Integrated transcriptomic and metabolomic analyses suggest various clusters of genes and metabolites are involved in distinct effects of HIIT, RT, and CT. These distinct metabolic signatures of different exercise modes independently link each type of exercise training to improved SI and cardiometabolic risk. ARTICLE HIGHLIGHTS: We aimed to understand the link between skeletal muscle metabolites and cardiometabolic health after exercise training. Although aerobic, resistance, and combined exercise training each enhance muscle insulin sensitivity as well as other cardiometabolic parameters, they disparately alter amino and citric acid metabolites as well as the lipidome, linking these metabolomic changes independently to the improvement of cardiometabolic risks with each exercise training mode. These findings reveal an important layer of the unique exercise mode-dependent changes in muscle metabolism, which may eventually lead to more informed exercise prescription for improving SI.

High-intensity aerobic, but not resistance or combined, exercise training improves both cardiometabolic health and skeletal muscle mitochondrial dynamics.

This study investigated how different exercise training modalities influence skeletal muscle mitochondrial dynamics. Healthy [average body mass index (BMI): 25.8 kg/m2], sedentary younger and older participants underwent 12 wk of supervised high-intensity aerobic interval training (HIIT; n = 13), resistance training (RT; n = 14), or combined training (CT; n = 11). Mitochondrial structure was assessed using transmission electron microscopy (TEM). Regulators of mitochondrial fission and fusion, cardiorespiratory fitness (V̇o2peak), insulin sensitivity via a hyperinsulinemic-euglycemic clamp, and muscle mitochondrial respiration were assessed. TEM showed increased mitochondrial volume, number, and perimeter following HIIT (P < 0.01), increased mitochondrial number following CT (P < 0.05), and no change in mitochondrial abundance after RT. Increased mitochondrial volume associated with increased mitochondrial respiration and insulin sensitivity following HIIT (P < 0.05). Increased mitochondrial perimeter associated with increased mitochondrial respiration, insulin sensitivity, and V̇o2peak following HIIT (P < 0.05). No such relationships were observed following CT or RT. OPA1, a regulator of fusion, was increased following HIIT (P < 0.05), whereas FIS1, a regulator of fission, was decreased following HIIT and CT (P < 0.05). HIIT also increased the ratio of OPA1/FIS1 (P < 0.01), indicative of the balance between fission and fusion, which positively correlated with improvements in respiration, insulin sensitivity, and V̇o2peak (P < 0.05). In conclusion, HIIT induces a larger, more fused mitochondrial tubular network. Changes indicative of increased fusion following HIIT associate with improvements in mitochondrial respiration, insulin sensitivity, and V̇o2peak supporting the idea that enhanced mitochondrial fusion accompanies notable health benefits of HIIT.NEW & NOTEWORTHY We assessed the effects of 12 wk of supervised high-intensity interval training (HIIT), resistance training, and combined training (CT) on skeletal muscle mitochondrial abundance and markers of fission and fusion. HIIT increased mitochondrial area and size and promoted protein changes indicative of increased mitochondrial fusion, whereas lessor effects were observed after CT and no changes were observed after RT. Furthermore, increased mitochondrial area and size after HIIT associated with improved mitochondrial respiration, cardiorespiratory fitness, and insulin sensitivity.

Impact of biological sex and sex hormones on molecular signatures of skeletal muscle at rest and in response to distinct exercise training modes.

Substantial divergence in cardio-metabolic risk, muscle size, and performance exists between men and women. Considering the pivotal role of skeletal muscle in human physiology, we investigated and found, based on RNA sequencing (RNA-seq), that differences in the muscle transcriptome between men and women are largely related to testosterone and estradiol and much less related to genes located on the Y chromosome. We demonstrate inherent unique, sex-dependent differences in muscle transcriptional responses to aerobic, resistance, and combined exercise training in young and older cohorts. The hormonal changes with age likely explain age-related differential expression of transcripts. Furthermore, in primary human myotubes we demonstrate the profound but distinct effects of testosterone and estradiol on amino acid incorporation to multiple individual proteins with specific functions. These results clearly highlight the potential of designing exercise programs tailored specifically to men and women and have implications for people who change gender by altering their hormone profile.

The Effect of Glucagon on Protein Catabolism During Insulin Deficiency: Exchange of Amino Acids Across Skeletal Muscle and the Splanchnic Bed.

Transient insulin deprivation with concurrent hyperglucagonemia is a catabolic state that can occur in type 1 diabetes. To evaluate glucagon's catabolic effect in the setting of its glucogenic effect, we measured the regional exchanges of amino acid metabolites (amino-metabolites) across muscle and splanchnic beds in 16 healthy humans during either somatostatin followed by glucagon or saline infusion alone. Despite a twofold or greater increase in the regional exchange of amino-metabolites by glucagon, whole-body kinetics and concentrations of amino acids (AA) remained stable. Glucagon increased the splanchnic uptake of not only gluconeogenic but also essential (EAA) AA while increasing their release from the muscle bed. Regional tracer-based kinetics and 3-methylhistidine release indicate that EAA release from muscle is likely caused by reduced protein synthesis rather than increased protein degradation. Furthermore, many metabolites known to affect insulin action and metabolism were altered by hyperglucagonemia including increase in branched-chain AA and keto acids of leucine and isoleucine in arterial plasma. Further, an increase in arterial concentrations of α-aminoadipic acid arising from increased conversion from lysine in the splanchnic bed was noted. These results demonstrate that hyperglucagonemia during hypoinsulinemia increases net muscle protein catabolism and substantially increases the exchange of amino metabolites across splanchnic and muscle beds.

Impaired Muscle Mitochondrial Function in Familial Partial Lipodystrophy.

CONTEXT: Familial partial lipodystrophy (FPL), Dunnigan variety is characterized by skeletal muscle hypertrophy and insulin resistance besides fat loss from the extremities. The cause for the muscle hypertrophy and its functional consequences is not known. OBJECTIVE: To compare muscle strength and endurance, besides muscle protein synthesis rate between subjects with FPL and matched controls (n = 6 in each group). In addition, we studied skeletal muscle mitochondrial function and gene expression pattern to help understand the mechanisms for the observed differences. METHODS: Body composition by dual-energy X-ray absorptiometry, insulin sensitivity by minimal modelling, assessment of peak muscle strength and fatigue, skeletal muscle biopsy and calculation of muscle protein synthesis rate, mitochondrial respirometry, skeletal muscle transcriptome, proteome, and gene set enrichment analysis. RESULTS: Despite increased muscularity, FPL subjects did not demonstrate increased muscle strength but had earlier fatigue on chest press exercise. Decreased mitochondrial state 3 respiration in the presence of fatty acid substrate was noted, concurrent to elevated muscle lactate and decreased long-chain acylcarnitine. Based on gene transcriptome, there was significant downregulation of many critical metabolic pathways involved in mitochondrial biogenesis and function. Moreover, the overall pattern of gene expression was indicative of accelerated aging in FPL subjects. A lower muscle protein synthesis and downregulation of gene transcripts involved in muscle protein catabolism was observed. CONCLUSION: Increased muscularity in FPL is not due to increased muscle protein synthesis and is likely due to reduced muscle protein degradation. Impaired mitochondrial function and altered gene expression likely explain the metabolic abnormalities and skeletal muscle dysfunction in FPL subjects.

Enhancement of anaerobic glycolysis - a role of PGC-1α4 in resistance exercise.

Resistance exercise training (RET) is an effective countermeasure to sarcopenia, related frailty and metabolic disorders. Here, we show that an RET-induced increase in PGC-1α4 (an isoform of the transcriptional co-activator PGC-1α) expression not only promotes muscle hypertrophy but also enhances glycolysis, providing a rapid supply of ATP for muscle contractions. In human skeletal muscle, PGC-1α4 binds to the nuclear receptor PPARß following RET, resulting in downstream effects on the expressions of key glycolytic genes. In myotubes, we show that PGC-1α4 overexpression increases anaerobic glycolysis in a PPARß-dependent manner and promotes muscle glucose uptake and fat oxidation. In contrast, we found that an acute resistance exercise bout activates glycolysis in an AMPK-dependent manner. These results provide a mechanistic link between RET and improved glucose metabolism, offering an important therapeutic target to counteract aging and inactivity-induced metabolic diseases benefitting those who cannot exercise due to many reasons.

Higher unacylated ghrelin and insulin sensitivity following dietary restriction and weight loss in obese humans.

BACKGROUND & AIMS: Unacylated ghrelin (UnAG) modulates insulin sensitivity. Low plasma UnAG occurs in obesity and potentially contributes to obesity-associated insulin resistance. We hypothesized that improvements in insulin sensitivity in obese people induced by moderate caloric restriction (CR) may be paralleled and at least in part explained by concurrent increases in UnAG levels. METHODS: 20 general community obese people were randomly assigned to 16-week CR (n = 11) or control diet (n = 9). We investigated the impact of CR on the interaction between insulin sensitivity changes [area under the curve (AUCg) of glucose infusion to maintain euglycemia during hyperinsulinemic-euglycemic clamp] and plasma total (TotalG), acylated (AG) and Unacylated ghrelin (UnAG). Plasma pro-inflammatory tumor necrosis factor alpha (TNFα) and anti-inflammatory interleukin-10 (IL-10) were also measured since changes in inflammation may contribute to UnAG activities. RESULTS: CR reduced BMI and increased insulin sensitivity (p < 0.05). TotalG and UnAG but not AG increased in CR but not in Control (p < 0.05). Il-10 and IL-10/TNFα ratio also increased in CR (p < 0.05). Changes in UnAG were positively associated with changes in AUCg in all subjects (n = 20; p < 0.01) also after adjustment for treatment and changes in BMI and cytokines. CONCLUSIONS: Caloric restriction modifies circulating ghrelin profile with selective increase in unacylated hormone in obese individuals. The current study supports the hypothesis that higher unacylated ghrelin contributes to improvements in insulin sensitivity following diet-induced weight loss in human obesity.

Transcriptomic Regulation of Muscle Mitochondria and Calcium Signaling by Insulin/IGF-1 Receptors Depends on FoxO Transcription Factors.

Insulin and IGF-1, acting through the insulin receptor (IR) and IGF-1 receptor (IGF1R), maintain muscle mass and mitochondrial function, at least part of which occurs via their action to regulate gene expression. Here, we show that while muscle-specific deletion of IR or IGF1R individually results in only modest changes in the muscle transcriptome, combined deletion of IR/IGF1R (MIGIRKO) altered > 3000 genes, including genes involved in mitochondrial dysfunction, fibrosis, cardiac hypertrophy, and pathways related to estrogen receptor, protein kinase A (PKA), and calcium signaling. Functionally, this was associated with decreased mitochondrial respiration and increased ROS production in MIGIRKO muscle. To determine the role of FoxOs in these changes, we performed RNA-Seq on mice with muscle-specific deletion of FoxO1/3/4 (M-FoxO TKO) or combined deletion of IR, IGF1R, and FoxO1/3/4 in a muscle quintuple knockout (M-QKO). This revealed that among IR/IGF1R regulated genes, >97% were FoxO-dependent, and their expression was normalized in M-FoxO TKO and M-QKO muscle. FoxO-dependent genes were related to oxidative phosphorylation, inflammatory signaling, and TCA cycle. Metabolomic analysis showed accumulation of TCA cycle metabolites in MIGIRKO, which was reversed in M-QKO muscle. Likewise, calcium signaling genes involved in PKA signaling and sarcoplasmic reticulum calcium homeostasis were markedly altered in MIGIRKO muscle but normalized in M-QKO. Thus, combined loss of insulin and IGF-1 action in muscle transcriptionally alters mitochondrial function and multiple regulatory and signaling pathways, and these changes are mediated by FoxO transcription factors.

Brain functions and cognition on transient insulin deprivation in type 1 diabetes.

BACKGROUNDType 1 diabetes (T1D) is a risk factor for dementia and structural brain changes. It remains to be determined whether transient insulin deprivation that frequently occurs in insulin-treated individuals with T1D alters brain function.METHODSWe therefore performed functional and structural magnetic resonance imaging, magnetic resonance spectroscopy, and neuropsychological testing at baseline and following 5.4 ± 0.6 hours of insulin deprivation in 14 individuals with T1D and compared results with those from 14 age-, sex-, and BMI-matched nondiabetic (ND) participants with no interventions.RESULTSInsulin deprivation in T1D increased blood glucose, and ß-hydroxybutyrate, while reducing bicarbonate levels. Participants with T1D showed lower baseline brain N-acetyl aspartate and myo-inositol levels but higher cortical fractional anisotropy, suggesting unhealthy neurons and brain microstructure. Although cognitive functions did not differ between participants with T1D and ND participants at baseline, significant changes in fine motor speed as well as attention and short-term memory occurred following insulin deprivation in participants with T1D. Insulin deprivation also reduced brain adenosine triphosphate levels and altered the phosphocreatine/adenosine triphosphate ratio. Baseline differences in functional connectivity in brain regions between participants with T1D and ND participants were noted, and on insulin deprivation further alterations in functional connectivity between regions, especially cortical and hippocampus-caudate regions, were observed. These alterations in functional connectivity correlated to brain metabolites and to changes in cognition.CONCLUSIONTransient insulin deprivation therefore caused alterations in executive aspects of cognitive function concurrent with functional connectivity between memory regions and the sensory cortex. These findings have important clinical implications, as many patients with T1D inadvertently have periods of transient insulin deprivation.TRIAL REGISTRATIONClinicalTrials.gov NCT03392441.FUNDINGClinical and Translational Science Award (UL1 TR002377) from the National Center for Advancing Translational Science; NIH grants (R21 AG60139 and R01 AG62859); the Mayo Foundation.

Higher muscle protein synthesis in women than men across the lifespan, and failure of androgen administration to amend age-related decrements.

We investigated age and sex effects and determined whether androgen replacement in elderly individuals (> or = 60 yr) could augment protein synthesis. Thirty young men and 32 young women (18-31 yr) were studied once, whereas 87 elderly men were studied before and after 1 yr of treatment with 5 mg/day testosterone (T), 75 mg/day dehydroepiandrosterone (DHEA), or placebo (P); and 57 elderly women were studied before and after 1 yr of treatment with 50 mg/day DHEA or P. [(15)N]Phenylalanine and [(2)H(4)]tyrosine tracers were infused, with measurements in plasma and vastus lateralis muscle. Whole-body protein synthesis per fat-free mass and muscle protein fractional synthesis rate (FSR) were lower in elderly than in young individuals (P<0.001), not significantly affected by hormone treatments, and higher in women than in men (P<0.0001), with no sex x age interaction. In regression analyses, peak O2 consumption (VO2peak), resting energy expenditure (REE), and sex were independently associated with muscle FSR, as were VO2peak, REE, and interactions of sex with insulin-like growth factor-II and insulin for whole-body protein synthesis. Women maintain higher protein synthesis than men across the lifespan as rates decline in both sexes, and neither full replacement of DHEA (in elderly men and women) nor partial replacement of bioavailable T (in elderly men) is able to amend the age-related declines.

Differential effects of insulin deprivation and systemic insulin treatment on plasma protein synthesis in type 1 diabetic people.

It remains to be determined whether systemic insulin replacement normalizes synthesis rates of different plasma proteins and whether there are differential effects on various plasma proteins. We tested a hypothesis that insulin deprivation differentially affects individual plasma protein synthesis and that systemic insulin treatment may not normalize synthesis of all plasma proteins. We measured synthesis rates of 41 plasma proteins in seven each of type 1 diabetic (T1DM) and nondiabetic participants (ND) using [ring-(13)C(6)]phenylalanine as a tracer. T1DM were studied while on chronic insulin treatment and during 8 h insulin deprivation. Insulin treatment normalized glucose levels, but plasma insulin levels were higher during insulin treatment than during insulin deprivation in T1DM and ND. Individual plasma proteins were purified by affinity chromatography and two-dimensional gel electrophoresis. Only 41 protein gel spots from over 300 were chosen based on their protein homogeneity. Insulin deprivation and hyperglycemia either significantly increased (n = 12) or decreased (n = 12) synthesis rates of 24 of 41 plasma proteins in T1DM compared with ND. Insulin treatment normalized synthesis rates of 13 of these 24 proteins, which were altered during insulin deprivation. However, insulin treatment significantly altered the synthesis of 14 additional proteins. In conclusion, acute insulin deprivation caused both a decrease and increase in synthesis rates of many plasma proteins with various functions. Moreover, chronic systemic insulin treatment not only did not normalize synthesis of all plasma proteins but also altered synthesis of several additional proteins that were unaltered during insulin deprivation.

Exercise and metformin counteract altered mitochondrial function in the insulin-resistant brain.

Insulin resistance associates with increased risk for cognitive decline and dementia; however, the underpinning mechanisms for this increased risk remain to be fully defined. As insulin resistance impairs mitochondrial oxidative metabolism and increases ROS in skeletal muscle, we considered whether similar events occur in the brain, which - like muscle - is rich in insulin receptors and mitochondria. We show that high-fat diet-induced (HFD-induced) brain insulin resistance in mice decreased mitochondrial ATP production rate and oxidative enzyme activities in brain regions rich in insulin receptors. HFD increased ROS emission and reduced antioxidant enzyme activities, with the concurrent accumulation of oxidatively damaged mitochondrial proteins and increased mitochondrial fission. Improvement of insulin sensitivity by both aerobic exercise and metformin ameliorated HFD-induced abnormalities. Moreover, insulin-induced enhancement of ATP production in primary cortical neurons and astrocytes was counteracted by the insulin receptor antagonist S961, demonstrating a direct effect of insulin resistance on brain mitochondria. Further, intranasal S961 administration prevented exercise-induced improvements in ATP production and ROS emission during HFD, supporting that exercise enhances brain mitochondrial function by improving insulin action. These results support that insulin sensitizing by exercise and metformin restores brain mitochondrial function in insulin-resistant states.

Mitochondrial Morphology, Dynamics, and Function in Human Pressure Overload or Ischemic Heart Disease With Preserved or Reduced Ejection Fraction.

BACKGROUND: The FOXO3a (forkhead box O3a)-BNIP3 (B-cell lymphoma 2/adenovirus E1B 19kDa interacting protein 3) pathway modulates mitochondrial dynamics and function and contributes to myocardial remodeling in rodent models of heart failure. We sought to investigate the expression of this pathway along with the expression of mitochondrial biogenesis (PGC-1α [peroxisome proliferator-activated receptor-γ coactivator-1α]), dynamics (DRP-1 [dynamin-related protein 1], OPA-1 [optic atrophy 1], and MFN 2 [mitofusin 2]), and oxidative phosphorylation (citrate synthase and electron transport chain complexes) markers and COX IV (cytochrome C oxidase) activity in myocardium from patients with valvular or ischemic heart disease and heart failure with preserved ejection fraction (HFpEF) or heart failure with reduced ejection fraction (HFrEF). METHODS AND RESULTS: Subepicardial left ventricular biopsies (10×1×1 mm3) were obtained at aortic valve replacement (HFpEFAVR, n=5; and HFrEFAVR, n=4), coronary artery bypass grafting (HFpEFCABG, n=5; and HFrEFCABG, n=5), or left ventricular assist device implantation (HFrEFLVAD, n=4). Subepicardial biopsies from patients with normal left ventricular function (n=2) and from donor hearts (n=3) served as controls (normal). Relative to normal, mitochondrial fragmentation and cristae destruction were evident, and mitochondrial area was decreased in HFpEF; 1.00±0.09 versus 0.71±0.08; P=0.016. These mitochondrial morphological changes were more pronounced in HFrEF (0.54±0.06); P=0.002 HFpEF versus HFrEF. BNIP3 (monomer+dimer) expression was increased in HFpEF (3.99±2.44) and in HFrEF (5.19±1.70) relative to normal; P=0.004 and P<0.001, respectively. However, BNIP3 monomer was increased in HFrEF (4.32±1.43) compared with normal (0.99±0.06) and HFpEF (1.97±0.90); P=0.001 and 0.004, respectively. The HFrEF group uniquely showed increase in DRP-1 expression (1.94±0.38) and decreases in PGC-1α expression (0.61±0.07) and COX IV activity (0.70±0.10) relative to normal; P=0.013, P<0.001, and P<0.001, respectively, with no significant change in electron transport chain complexes expression. CONCLUSIONS: These findings in human myocardium confirm studies in rodents where contractile dysfunction is associated with activation of the FOXO3a-BNIP3 pathway and altered mitochondrial dynamics, biogenesis, and function.

FoxO Transcription Factors Are Critical Regulators of Diabetes-Related Muscle Atrophy.

Insulin deficiency and uncontrolled diabetes lead to a catabolic state with decreased muscle strength, contributing to disease-related morbidity. FoxO transcription factors are suppressed by insulin and thus are key mediators of insulin action. To study their role in diabetic muscle wasting, we created mice with muscle-specific triple knockout of FoxO1/3/4 and induced diabetes in these M-FoxO-TKO mice with streptozotocin (STZ). Muscle mass and myofiber area were decreased 20-30% in STZ-Diabetes mice due to increased ubiquitin-proteasome degradation and autophagy alterations, characterized by increased LC3-containing vesicles, and elevated levels of phosphorylated ULK1 and LC3-II. Both the muscle loss and markers of increased degradation/autophagy were completely prevented in STZ FoxO-TKO mice. Transcriptomic analyses revealed FoxO-dependent increases in ubiquitin-mediated proteolysis pathways in STZ-Diabetes, including regulation of Fbxo32 (Atrogin1), Trim63 (MuRF1), Bnip3L, and Gabarapl. These same genes were increased 1.4- to 3.3-fold in muscle from humans with type 1 diabetes after short-term insulin deprivation. Thus, FoxO-regulated genes play a rate-limiting role in increased protein degradation and muscle atrophy in insulin-deficient diabetes.

Influence of omega-3 fatty acids on skeletal muscle protein metabolism and mitochondrial bioenergetics in older adults.

Omega-3 polyunsaturated fatty acids (n3-PUFA) are recognized for their anti-inflammatory effects and may be beneficial in the context of sarcopenia. We determined the influence of n3-PUFA on muscle mitochondrial physiology and protein metabolism in older adults. Twelve young (18-35 years) and older (65-85 years) men and women were studied at baseline. Older adults were studied again following n3-PUFA supplementation (3.9g/day, 16 weeks). Muscle biopsies were used to evaluate respiratory capacity (high resolution respirometry) and oxidant emissions (spectrofluorometry) in isolated mitochondria. Maximal respiration was significantly lower in older compared to young. n3-PUFA did not change respiration, but significantly reduced oxidant emissions. Participants performed a single bout of resistance exercise, followed by biopsies at 15 and 18 hours post exercise. Several genes involved in muscle protein turnover were significantly altered in older adults at baseline and following exercise, yet muscle protein synthesis was similar between age groups under both conditions. Following n3-PUFA supplementation, mixed muscle, mitochondrial, and sarcoplasmic protein synthesis rates were increased in older adults before exercise. n3-PUFA increased post-exercise mitochondrial and myofibrillar protein synthesis in older adults. These results demonstrate that n3-PUFA reduce mitochondrial oxidant emissions, increase postabsorptive muscle protein synthesis, and enhance anabolic responses to exercise in older adults.