Phillip J. White

Long-chain omega-3 fatty acids regulate bovine whole-body protein metabolism by promoting muscle insulin signalling to the Akt–mTOR–S6K1 pathway and insulin sensitivity

The ability of the skeletal musculature to use amino acids to build or renew constitutive proteins is gradually lost with age and this is partly due to a decline in skeletal muscle insulin sensitivity. Since long‐chain omega‐3 polyunsaturated fatty acids (LCn–3PUFA) from fish oil are known to improve insulin‐mediated glucose metabolism in insulin‐resistant states, their potential role in regulating insulin‐mediated protein metabolism was investigated in this study. Experimental data are based on a switchback design composed of three 5 week experimental periods using six growing steers to compare the effect of a continuous abomasal infusion of LCn–3PUFA‐rich menhaden oil with an iso‐energetic control oil mixture. Clamp and insulin signalling observations were combined with additional data from a second cohort of six steers. We found that enteral LCn–3PUFA potentiate insulin action by increasing the insulin‐stimulated whole‐body disposal of amino acids from 152 to 308 μmol kg−1 h−1 (P= 0.006). The study further showed that in the fed steady‐state, chronic adaptation to LCn–3PUFA induces greater activation (P < 0.05) of the Akt–mTOR–S6K1 signalling pathway. Simultaneously, whole‐body total flux of phenylalanine was reduced from 87 to 67 μmol kg−1 h−1 (P= 0.04) and oxidative metabolism was decreased (P= 0.05). We conclude that chronic feeding of menhaden oil provides a novel nutritional mean to enhance insulin‐sensitive aspects of protein metabolism.

Transgenic Restoration of Long-Chain n-3 Fatty Acids in Insulin Target Tissues Improves Resolution Capacity and Alleviates Obesity-Linked Inflammation and Insulin Resistance in High-Fat–Fed Mice

OBJECTIVE The catabasis of inflammation is an active process directed by n-3 derived pro-resolving lipid mediators. We aimed to determine whether high-fat (HF) diet-induced n-3 deficiency compromises the resolution capacity of obese mice and thereby contributes to obesity-linked inflammation and insulin resistance. RESEARCH DESIGN AND METHODS We used transgenic expression of the fat-1 n-3 fatty acid desaturase from C. elegans to endogenously restore n-3 fatty acids in HF-fed mice. After 8 weeks on HF or chow diets, wild-type and fat-1 transgenic mice were subjected to insulin and glucose tolerance tests and a resolution assay was performed. Metabolic tissues were then harvested for biochemical analyses. RESULTS We report that the n-3 docosanoid resolution mediator protectin D1 is lacking in muscle and adipose tissue of HF-fed wild-type mice. Accordingly, HF-fed wild-type mice have an impaired capacity to resolve an acute inflammatory response and display elevated adipose macrophage accrual and chemokine/cytokine expression. This is associated with insulin resistance and higher activation of iNOS and JNK in muscle and liver. These defects are reversed in HF-fed fat-1 mice, in which the biosynthesis of this important n-3 docosanoid resolution mediator is improved. Importantly, transgenic restoration of n-3 fatty acids prevented obesity-linked inflammation and insulin resistance in HF-fed mice without altering food intake, weight gain, or adiposity. CONCLUSIONS We conclude that inefficient biosynthesis of n-3 resolution mediators in muscle and adipose tissue contributes to the maintenance of chronic inflammation in obesity and that these novel lipids offer exciting potential for the treatment of insulin resistance and diabetes.

Insulin Reverses the High-Fat Diet–Induced Increase in Brain Aβ and Improves Memory in an Animal Model of Alzheimer Disease

Defects in insulin production and signaling are suspected to share a key role in diabetes and Alzheimer disease (AD), two age-related pathologies. In this study, we investigated the interrelation between AD and diabetes using a high-fat diet (HFD) in a mouse model of genetically induced AD-like neuropathology (3xTg-AD). We first observed that cerebral expression of human AD transgenes led to peripheral glucose intolerance, associated with pancreatic human Aβ accumulation. High-fat diet enhanced glucose intolerance, brain soluble Aβ, and memory impairment in 3xTg-AD mice. Strikingly, a single insulin injection reversed the deleterious effects of HFD on memory and soluble Aβ levels, partly through changes in Aβ production and/or clearance. Our results are consistent with the development of a vicious cycle between AD and diabetes, potentiating both peripheral metabolic disorders and AD neuropathology. The capacity of insulin to rapidly break the deleterious effects of this cycle on soluble Aβ concentrations and memory has important therapeutic implications.

Differential effects of various fish proteins in altering body weight, adiposity, inflammatory status, and insulin sensitivity in high-fat-fed rats.

Mounting evidence suggests that the benefits of fish consumption are not limited to the well-appreciated effects of omega-3 fatty acids. We previously demonstrated that cod protein protects against the development of diet-induced insulin resistance. The goal of this study was to determine whether other fish protein sources present similar beneficial effects. Rats were fed a high-fat, high-sucrose diet containing protein from casein or fish proteins from bonito, herring, mackerel, or salmon. After 28 days, oral glucose tolerance tests or hyperinsulinemic-euglycemic clamps were performed; and tissues and plasma were harvested for biochemical analyses. Despite equal energy intake among all groups, the salmon-protein-fed group presented significantly lower weight gain that was associated with reduced fat accrual in epididymal white adipose tissue. Although this reduction in visceral adiposity was not associated with improved glucose tolerance, we found that whole-body insulin sensitivity for glucose metabolism was improved using the very sensitive hyperinsulinemic-euglycemic clamp technique. Importantly, expression of both tumor necrosis factor-α and interleukin-6 was reduced in visceral adipose tissue of all fish-protein-fed groups when compared with the casein-fed control group, suggesting that fish proteins carry anti-inflammatory properties that may protect against obesity-linked metabolic complications. Interestingly, consumption of the salmon protein diet was also found to raise circulating salmon calcitonin levels, which may underlie the reduction of weight gain in these rats. These data suggest that not all fish protein sources exert the same beneficial properties on the metabolic syndrome, although anti-inflammatory actions appear to be common.

Protectin DX alleviates insulin resistance by activating a myokine-liver glucoregulatory axis

We previously demonstrated that low biosynthesis of ω–3 fatty acid–derived proresolution mediators, termed protectins, is associated with an impaired global resolution capacity, inflammation and insulin resistance in obese high-fat diet–fed mice. These findings prompted a more direct study of the therapeutic potential of protectins for the treatment of metabolic disorders. Herein we show that protectin DX (PDX) exerts an unanticipated glucoregulatory activity that is distinct from its anti-inflammatory actions. We found that PDX selectively stimulated the release of the prototypic myokine interleukin-6 (IL-6) from skeletal muscle and thereby initiated a myokine-liver signaling axis, which blunted hepatic glucose production via signal transducer and activator of transcription 3 (STAT3)-mediated transcriptional suppression of the gluconeogenic program. These effects of PDX were abrogated in Il6-null mice. PDX also activated AMP-activated protein kinase (AMPK); however, it did so in an IL-6–independent manner. Notably, we demonstrated that administration of PDX to obese diabetic db/db mice raises skeletal muscle IL-6 levels and substantially improves their insulin sensitivity without any impact on adipose tissue inflammation. Our findings thus support the development of PDX-based selective muscle IL-6 secretagogues as a new class of therapy for the treatment of insulin resistance and type 2 diabetes.

Endotoxin mediated-iNOS induction causes insulin resistance via ONOO⁻ induced tyrosine nitration of IRS-1 in skeletal muscle.

Background It is believed that the endotoxin lipopolysaccharide (LPS) is implicated in the metabolic perturbations associated with both sepsis and obesity (metabolic endotoxemia). Here we examined the role of inducible nitric oxide synthase (iNOS) in skeletal muscle insulin resistance using LPS challenge in rats and mice as in vivo models of endotoxemia. Methodology/Principal Findings Pharmacological (aminoguanidine) and genetic strategies (iNOS−/− mice) were used to counter iNOS induction in vivo. In vitro studies using peroxynitrite (ONOO−) or inhibitors of the iNOS pathway, 1400 W and EGCG were conducted in L6 myocytes to determine the mechanism by which iNOS mediates LPS-dependent insulin resistance. In vivo, both pharmacological and genetic invalidation of iNOS prevented LPS-induced muscle insulin resistance. Inhibition of iNOS also prevented insulin resistance in myocytes exposed to cytokine/LPS while exposure of myocytes to ONOO− fully reproduced the inhibitory effect of cytokine/LPS on both insulin-stimulated glucose uptake and PI3K activity. Importantly, LPS treatment in vivo and iNOS induction and ONOO− treatment in vitro promoted tyrosine nitration of IRS-1 and reduced insulin-dependent tyrosine phosphorylation. Conclusions/Significance Our work demonstrates that iNOS-mediated tyrosine nitration of IRS-1 is a key mechanism of skeletal muscle insulin resistance in endotoxemia, and presents nitrosative modification of insulin signaling proteins as a novel therapeutic target for combating muscle insulin resistance in inflammatory settings.

Branched-chain amino acid restriction in Zucker-fatty rats improves muscle insulin sensitivity by enhancing efficiency of fatty acid oxidation and acyl-glycine export

Objective A branched-chain amino acid (BCAA)-related metabolic signature is strongly associated with insulin resistance and predictive of incident diabetes and intervention outcomes. To better understand the role that this metabolite cluster plays in obesity-related metabolic dysfunction, we studied the impact of BCAA restriction in a rodent model of obesity in which BCAA metabolism is perturbed in ways that mirror the human condition. Methods Zucker-lean rats (ZLR) and Zucker-fatty rats (ZFR) were fed either a custom control, low fat (LF) diet, or an isonitrogenous, isocaloric LF diet in which all three BCAA (Leu, Ile, Val) were reduced by 45% (LF-RES). We performed comprehensive metabolic and physiologic profiling to characterize the effects of BCAA restriction on energy balance, insulin sensitivity, and glucose, lipid and amino acid metabolism. Results LF-fed ZFR had higher levels of circulating BCAA and lower levels of glycine compared to LF-fed ZLR. Feeding ZFR with the LF-RES diet lowered circulating BCAA to levels found in LF-fed ZLR. Activity of the rate limiting enzyme in the BCAA catabolic pathway, branched chain keto acid dehydrogenase (BCKDH), was lower in liver but higher in skeletal muscle of ZFR compared to ZLR and was not responsive to diet in either tissue. BCAA restriction had very little impact on metabolites studied in liver of ZFR where BCAA content was low, and BCKDH activity was suppressed. However, in skeletal muscle of LF-fed ZFR compared to LF-fed ZLR, where BCAA content and BCKDH activity were increased, accumulation of fatty acyl CoAs was completely normalized by dietary BCAA restriction. BCAA restriction also normalized skeletal muscle glycine content and increased urinary acetyl glycine excretion in ZFR. These effects were accompanied by lower RER and improved skeletal muscle insulin sensitivity in LF-RES fed ZFR as measured by hyperinsulinemic-isoglycemic clamp. Conclusions Our data are consistent with a model wherein elevated circulating BCAA contribute to development of obesity-related insulin resistance by interfering with lipid oxidation in skeletal muscle. BCAA-dependent lowering of the skeletal muscle glycine pool appears to contribute to this effect by slowing acyl-glycine export to the urine.

Transgenic ω-3 PUFA enrichment alters morphology and gene expression profile in adipose tissue of obese mice: Potential role for protectins

OBJECTIVE Dietary administration of ω-3 polyunsaturated fatty acids (PUFA) is often associated with altered adipose tissue (AT) morphology and/or function in obese mice. Yet, it is unclear whether this is an indirect consequence of reduced weight gain or results from direct actions of ω-3 PUFA. Here we studied the AT of high fat (HF)-fed fat-1 transgenic mice that convert endogenous ω-6 to ω-3 PUFA while maintaining equivalent fat accretion as their wild-type (WT) counterparts. MATERIALS AND METHODS Adipocyte size profiling, Affymetrix microarray pathway analysis, qPCR and protectin identification and analysis were performed in epididymal AT from hemizygous fat-1(+/-) mice and their wild type littermates that had been fed a HF diet for 8weeks from 6weeks of age. RESULTS Despite equivalent fat pad mass, we found that epididymal AT from HF-fed transgenic animals possesses fewer large and very large but more mid-size adipocytes compared to WT mice. In order to better understand the underlying mechanisms contributing to the observed alteration in adipocyte size we performed an Affymetrix microarray. Pathway analysis of these data highlighted adipogenesis, cholesterol biosynthesis, insulin signaling, prostaglandin synthesis/regulation and small ligand GPCRs as points where differentially expressed genes were significantly overrepresented. Observed changes were confirmed for four candidate genes: Cnr1, Cnr2, Faah and Pparg by qPCR. Finally we demonstrated that protectin DX is present in AT and that protectin DX and protectin D1 promote comparable PPARγ transcriptional activity. CONCLUSIONS These data provide unprecedented evidence that ω-3 PUFA coordinately regulate AT gene expression programs in a manner that is independent of restriction of weight gain or fat accrual and highlight an important influence of ω-3 PUFA on adipogenesis. Furthermore we provide primary evidence suggesting that protectins likely contribute to these effects via their influence on PPARγ.

Is omega-3 key to unlocking inflammation in obesity?

Low-grade inflammation has been identified as a key player in the development of the metabolic syndrome in obese subjects, leading the way to type 2 diabetes and cardiovascular diseases. Insulin resistance, a primary component of the metabolic syndrome, is characterised by chronically elevated concentrations of proinflammatory cytokines, acute phase proteins, and enhanced activation of proinflammatory signalling in insulin-responsive tissues of obese subjects. The research effort thus far has established that, in obesity, the expanding adipose tissue makes a substantial contribution to the development of obesity-linked inflammation via the secretion of various proinflammatory cytokines, chemokines and adipokines. Recent investigations into the mechanisms responsible for this phenomenon have outlined a role for the macrophage [1–3]. In fact, macrophage accumulation in adipose tissue is positively correlated with adipocyte size and contributes to the expression of proinflammatory mediators of insulin resistance, such as TNF-α, IL-6 and inducible nitric oxide synthase (iNOS) [1, 2]. Furthermore, both inhibition of macrophage function, via myeloid-specific IκB kinase-β deletion [3], and prevention of macrophage accumulation in adipose tissue, via deletion of C–C motif chemokine receptor-2 [4], prevent the development of the inflammatory phenotype and insulin resistance with obesity. Hence, there is growing support for the current notion that macrophage infiltration into adipose tissue is central to obesity-related metabolic disorders. Pioneering work by Storlien and colleagues has shown that long-chain omega-3 polyunsaturated fatty acids (LCn− 3PUFA) can prevent the development of diet-induced insulin resistance in rats [5]. Furthermore, a growing number of studies support a link between this beneficial effect of LCn−3PUFA and an anti-inflammatory mechanism. LCn−3PUFA have been widely reported to have antiinflammatory effects in a range of chronic inflammatory conditions, including rheumatoid arthritis and Crohn’s disease [6, 7]. Treatment of obese subjects with LCn− 3PUFA in a clinical setting has also been shown to reduce circulating levels of both proinflammatory cytokines and acute phase proteins [8, 9]. Whether these anti-inflammatory actions of LCn−3PUFA, and their positive influence on the metabolic syndrome, can be linked to local blunting of adipose tissue inflammation is not yet known. In this issue of Diabetologia, Todoric et al. [10] present evidence that the inclusion of LCn−3PUFA in a high-fat diet prevents the development of an inflammatory gene expression profile and macrophage infiltration in the adipose tissue of obese diabetic db/db mice. Despite significantly enhancing weight gain, LCn−3PUFA treatment completely prevented the diet-induced adipose tissue switch to an inflammatory profile, attenuating the upregulation of an array of genes, notably those encoding the macrophage surface marker CD68, macrophage chemotactic protein-1, and the lipopolysaccharide receptor CD14. Incorporation of LCn−3PUFA into the high-fat diet also prevented the downregulation of genes involved in lipid metabolism, including those for fatty acid synthase and hormone-sensitive lipase, in adipose tissue of the obese mice. The anti-inflammatory effect of LCn−3PUFA was related to greatly diminished macrophage migration into Diabetologia (2006) 49:1999–2001 DOI 10.1007/s00125-006-0346-9

Age-dependent impairment of glucose tolerance in the 3xTg-AD mouse model of Alzheimer’s disease

Alzheimers disease (AD) has been associated with type II diabetes (T2D) and obesity in several epidemiologic studies. To determine whether AD neuropathology can cause peripheral metabolic impairments, we investigated metabolic parameters in the triple‐transgenic (3xTg)‐AD mouse model of AD, compared with those in nontransgenic (non‐Tg) controls, at 6, 8, and 14 mo of age. We found a more pronounced cortical Aβ accumulation (2‐and 3.5‐fold increase in Aβ42 in the soluble and insoluble protein fractions, respectively) in female 3xTg‐AD mice than in the males. Furthermore, female 3xTg‐AD mice displayed a significant deterioration in glucose tolerance (AUC, +118% vs. non‐Tg mice at 14 mo). Fasting plasma insulin levels rose 2.5‐fold from 6 to 14 mo of age in female 3xTg‐AD mice. Glucose intolerance and cortical amyloid pathology worsened with age, and both were more pronounced in the females. Pancreatic amyloidopathy was revealed and could underlie the observed deficit in glycemic response in 3xTg‐AD mice. The present results suggest that AD‐like neuropathology extends to the pancreas in the 3xTg‐AD mouse, leading to glucose intolerance and contributing to a pathologic self‐amplifying loop between AD and T2D.—Vandal, M., White, P. J., Chevrier, G., Tremblay, C., St‐Amour, I., Planel, E., Marette, A., Calon, F. Age‐dependent impairment of glucose tolerance in the 3xTg‐AD mouse model of Alzheimers disease. FASEB J. 29, 4273‐4284 (2015). www.fasebj.org