Cardiac arrest (CA) causes high mortality due to multi-system organ damage attributable to ischemia-reperfusion injury. Recent work in our group found that among diabetic patients who experienced cardiac arrest, those taking metformin had less evidence of cardiac and renal damage after cardiac arrest when compared to those not taking metformin. Based on these observations, we hypothesized that metformin's protective effects in the heart were mediated by AMPK signaling, and that AMPK signaling could be targeted as a therapeutic strategy following resuscitation from CA. The current study investigates metformin interventions on cardiac and renal outcomes in a non-diabetic CA mouse model. We found that two weeks of metformin pretreatment protects against reduced ejection fraction and reduces kidney ischemia-reperfusion injury at 24 h post-arrest. This cardiac and renal protection depends on AMPK signaling, as demonstrated by outcomes in mice pretreated with the AMPK activator AICAR or metformin plus the AMPK inhibitor compound C. At this 24-h time point, heart gene expression analysis showed that metformin pretreatment caused changes supporting autophagy, antioxidant response, and protein translation. Further investigation found associated improvements in mitochondrial structure and markers of autophagy. Notably, Western analysis indicated that protein synthesis was preserved in arrest hearts of animals pretreated with metformin. The AMPK activation-mediated preservation of protein synthesis was also observed in a hypoxia/reoxygenation cell culture model. Despite the positive impacts of pretreatment in vivo and in vitro, metformin did not preserve ejection fraction when deployed at resuscitation. Taken together, we propose that metformin's in vivo cardiac preservation occurs through AMPK activation, requires adaptation before arrest, and is associated with preserved protein translation.

The brain is the most cholesterol-rich organ in the body, most of which comes from in situ synthesis. Here we demonstrate that in insulin-deficient diabetic mice, there is a reduction in expression of the major transcriptional regulator of cholesterol metabolism, SREBP-2, and its downstream genes in the hypothalamus and other areas of the brain, leading to a reduction in brain cholesterol synthesis and synaptosomal cholesterol content. These changes are due, at least in part, to direct effects of insulin to regulate these genes in neurons and glial cells and can be corrected by intracerebroventricular injections of insulin. Knockdown of SREBP-2 in cultured neurons causes a decrease in markers of synapse formation and reduction of SREBP-2 in the hypothalamus of mice using shRNA results in increased feeding and weight gain. Thus, insulin and diabetes can alter brain cholesterol metabolism, and this may play an important role in the neurologic and metabolic dysfunction observed in diabetes and other disease states.

Body weight is regulated by coordinating energy intake and energy expenditure. Transforming growth factor β (TGFβ)/bone morphogenetic protein (BMP) signaling has been shown to regulate energy balance in lower organisms, but whether a similar pathway exists in mammals is unknown. We have previously demonstrated that BMP7 can regulate brown adipogenesis and energy expenditure. In the current study, we have uncovered a novel role for BMP7 in appetite regulation. Systemic treatment of diet-induced obese mice with BMP7 resulted in increased energy expenditure and decreased food intake, leading to a significant reduction in body weight and improvement of metabolic syndrome. Similar degrees of weight loss with reduced appetite were also observed in BMP7-treated ob/ob mice, suggesting a leptin-independent mechanism utilized by BMP7. Intracerebroventricular administration of BMP7 to mice led to an acute decrease in food intake, which was mediated, at least in part, by a central rapamycin-sensitive mTOR-p70S6 kinase pathway. Together, these results underscore the importance of BMP7 in regulating both food intake and energy expenditure, and suggest new therapeutic approaches for obesity and its comorbidities.

Inhibition of Hsp90 has been shown to improve glucose tolerance and insulin sensitivity in mouse models of diabetes. In the present report, the specific isoform Hsp90ab1, was identified as playing a major role in regulating insulin signaling and glucose metabolism.

Sirtuins are NAD(+)-dependent protein deacetylases. They mediate adaptive responses to a variety of stresses, including calorie restriction and metabolic stress. Sirtuin 3 (SIRT3) is localized in the mitochondrial matrix, where it regulates the acetylation levels of metabolic enzymes, including acetyl coenzyme A synthetase 2 (refs 1, 2). Mice lacking both Sirt3 alleles appear phenotypically normal under basal conditions, but show marked hyperacetylation of several mitochondrial proteins. Here we report that SIRT3 expression is upregulated during fasting in liver and brown adipose tissues. During fasting, livers from mice lacking SIRT3 had higher levels of fatty-acid oxidation intermediate products and triglycerides, associated with decreased levels of fatty-acid oxidation, compared to livers from wild-type mice. Mass spectrometry of mitochondrial proteins shows that long-chain acyl coenzyme A dehydrogenase (LCAD) is hyperacetylated at lysine 42 in the absence of SIRT3. LCAD is deacetylated in wild-type mice under fasted conditions and by SIRT3 in vitro and in vivo; and hyperacetylation of LCAD reduces its enzymatic activity. Mice lacking SIRT3 exhibit hallmarks of fatty-acid oxidation disorders during fasting, including reduced ATP levels and intolerance to cold exposure. These findings identify acetylation as a novel regulatory mechanism for mitochondrial fatty-acid oxidation and demonstrate that SIRT3 modulates mitochondrial intermediary metabolism and fatty-acid use during fasting.

Despite the well-documented association between gallstones and the metabolic syndrome, the mechanistic links between these two disorders remain unknown. Here we show that mice solely with hepatic insulin resistance, created by liver-specific disruption of the insulin receptor (LIRKO mice) are markedly predisposed toward cholesterol gallstone formation due to at least two distinct mechanisms. Disinhibition of the forkhead transcription factor FoxO1, increases expression of the biliary cholesterol transporters Abcg5 and Abcg8, resulting in an increase in biliary cholesterol secretion. Hepatic insulin resistance also decreases expression of the bile acid synthetic enzymes, particularly Cyp7b1, and produces partial resistance to the farnesoid X receptor, leading to a lithogenic bile salt profile. As a result, after twelve weeks on a lithogenic diet, all of the LIRKO mice develop gallstones. Thus, hepatic insulin resistance provides a crucial link between the metabolic syndrome and increased cholesterol gallstone susceptibility.

While most monogenic diseases are caused by loss or reduction of protein function, the need for technologies that can selectively increase levels of protein in native tissues remains. Here we demonstrate that antisense-mediated modulation of pre-mRNA splicing can increase endogenous expression of full-length protein by preventing naturally occurring non-productive alternative splicing and promoting generation of productive mRNA. Bioinformatics analysis of RNA sequencing data identifies non-productive splicing events in 7,757 protein-coding human genes, of which 1,246 are disease-associated. Antisense oligonucleotides targeting multiple types of non-productive splicing events lead to increases in productive mRNA and protein in a dose-dependent manner in vitro. Moreover, intracerebroventricular injection of two antisense oligonucleotides in wild-type mice leads to a dose-dependent increase in productive mRNA and protein in the brain. The targeting of natural non-productive alternative splicing to upregulate expression from wild-type or hypomorphic alleles provides a unique approach to treating genetic diseases.

Sirt3 is an NAD(+)-dependent deacetylase that regulates mitochondrial function by targeting metabolic enzymes and proteins. In fasting mice, Sirt3 expression is decreased in skeletal muscle resulting in increased mitochondrial protein acetylation. Deletion of Sirt3 led to impaired glucose oxidation in muscle, which was associated with decreased pyruvate dehydrogenase (PDH) activity, accumulation of pyruvate and lactate metabolites, and an inability of insulin to suppress fatty acid oxidation. Antibody-based acetyl-peptide enrichment and mass spectrometry of mitochondrial lysates from WT and Sirt3 KO skeletal muscle revealed that a major target of Sirt3 deacetylation is the E1α subunit of PDH (PDH E1α). Sirt3 knockout in vivo and Sirt3 knockdown in myoblasts in vitro induced hyperacetylation of the PDH E1α subunit, altering its phosphorylation leading to suppressed PDH enzymatic activity. The inhibition of PDH activity resulting from reduced levels of Sirt3 induces a switch of skeletal muscle substrate utilization from carbohydrate oxidation toward lactate production and fatty acid utilization even in the fed state, contributing to a loss of metabolic flexibility. Thus, Sirt3 plays an important role in skeletal muscle mitochondrial substrate choice and metabolic flexibility in part by regulating PDH function through deacetylation.

Excessive production of triglyceride-rich very low-density lipoproteins (VLDL-TG) contributes to hypertriglyceridemia in obesity and type 2 diabetes. To understand the underlying mechanism, we studied hepatic regulation of VLDL-TG production by (forkhead box O6) FoxO6, a forkhead transcription factor that integrates insulin signaling to hepatic metabolism. We showed that transgenic mice expressing a constitutively active FoxO6 allele developed hypertriglyceridemia, culminating in elevated VLDL-TG levels and impaired postprandial TG clearance. This effect resulted in part from increased hepatic VLDL-TG production. We recapitulated these findings in cultured HepG2 cells and human primary hepatocytes, demonstrating that FoxO6 promoted hepatic VLDL-TG secretion. This action correlated with the ability of FoxO6 to stimulate hepatic production of microsomal triglyceride transfer protein (MTP), a molecular chaperone that catalyzes the rate-limiting step in VLDL-TG assembly and secretion. FoxO6 was shown to bind to the MTP promoter and stimulate MTP promoter activity in HepG2 cells. This effect was inhibited by insulin, consistent with the ability of insulin to promote FoxO6 phosphorylation and disable FoxO6 DNA-binding activity. Mutations of the FoxO6 target site within the MTP promoter abrogated FoxO6-mediated induction of MTP promoter activity. Hepatic FoxO6 expression became deregulated in insulin-resistant mice with obesity and type 2 diabetes. FoxO6 inhibition in insulin-resistant liver suppressed hepatic MTP expression and curbed VLDL-TG overproduction, contributing to the amelioration of hypertriglyceridemia in obese and diabetic db/db mice. These results characterize FoxO6 as an important signaling molecule upstream of MTP for regulating hepatic VLDL-TG production.

Mitochondrial trifunctional protein (TFP) is a membrane-associated heterotetramer that catalyzes three of the four reactions needed to chain-shorten long-chain fatty acids inside the mitochondria. TFP is known to be heavily modified by acetyllysine and succinyllysine post-translational modifications (PTMs), many of which are targeted for reversal by the mitochondrial sirtuin deacylases SIRT3 and SIRT5. However, the functional significance of these PTMs is not clear, with some reports showing TFP gain-of-function and some showing loss-of-function upon increased acylation. Here, we mapped the known SIRT3/SIRT5-targeted lysine residues onto the recently solved TFP crystal structure which revealed that many of the target sites are involved in substrate channeling within the TFPα subunit. To test the effects of acylation on substate channeling through TFPα, we enzymatically synthesized the physiological long-chain substrate (2E)-hexadecenoyl-CoA. Assaying TFP in SIRT3 and SIRT5 knockout mouse liver and heart mitochondria with (2E)-hexadecenoyl-CoA revealed no change in enzyme activity. Finally, we investigated the effects of lysine acylation on TFP membrane binding in vitro. Acylation did not alter recombinant TFP binding to cardiolipin-containing liposomes. However, the presence of liposomes strongly abrogated the acylation reaction between succinyl-CoA and TFP lysine residues. Thus, TFP in the membrane-bound state may be protected against lysine acylation.

Acetylation is increasingly recognized as an important metabolic regulatory posttranslational protein modification, yet the metabolic consequence of mitochondrial protein hyperacetylation is unknown. We find that high-fat diet (HFD) feeding induces hepatic mitochondrial protein hyperacetylation in mice and downregulation of the major mitochondrial protein deacetylase SIRT3. Mice lacking SIRT3 (SIRT3KO) placed on a HFD show accelerated obesity, insulin resistance, hyperlipidemia, and steatohepatitis compared to wild-type (WT) mice. The lipogenic enzyme stearoyl-CoA desaturase 1 is highly induced in SIRT3KO mice, and its deletion rescues both WT and SIRT3KO mice from HFD-induced hepatic steatosis and insulin resistance. We further identify a single nucleotide polymorphism in the human SIRT3 gene that is suggestive of a genetic association with the metabolic syndrome. This polymorphism encodes a point mutation in the SIRT3 protein, which reduces its overall enzymatic efficiency. Our findings show that loss of SIRT3 and dysregulation of mitochondrial protein acetylation contribute to the metabolic syndrome.

Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved label-free approaches. This need is particularly pressing for workflows that incorporate affinity enrichment at the peptide level, where isobaric chemical labels such as isobaric tags for relative and absolute quantitation and tandem mass tags may prove problematic or where stable isotope labeling with amino acids in cell culture labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from full scan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to ensure proper peak selection. The modular structure of Skyline also provides well defined, customizable data reports and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 filtering approach, we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important post-translational modifications: acetylation and phosphorylation, in peptide-centric affinity workflows of increasing complexity using mouse and human models.