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Microvascular insulin delivery to myocytes is rate limiting for the onset of insulin-stimulated muscle glucose uptake. The structural integrity of capillaries of the microvasculature is regulated, in part, by a family of transmembrane adhesion receptors known as integrins, which are composed of an α and a β subunit. The integrin β1 (itgβ1) subunit is highly expressed in endothelial cells (ECs). EC itgβ1 is necessary for the formation of capillary networks during embryonic development, and its knockdown in adult mice blunts the reactive hyperemia that manifests during ischemia reperfusion. In this study, we investigated the contribution of EC itgβ1 in microcirculatory function and glucose uptake, with an emphasis on skeletal muscle. We hypothesized that loss of EC itgβ1 would impair microvascular hemodynamics and glucose uptake during insulin stimulation, creating "delivery"-mediated insulin resistance. An itgβ1 knockdown mouse model was developed to avoid the lethality of embryonic gene knockout and the deteriorating health resulting from early postnatal inducible gene deletion. We found that mice with ( itgβ1 fl/fl SCLcre) and without ( itgβ1 fl/fl ) inducible stem cell leukemia cre recombinase (SLCcre) expression at 10 days post cre induction have comparable exercise tolerance and pulmonary and cardiac functions. We quantified microcirculatory hemodynamics using intravital microscopy and the ability of mice to respond to the high metabolic demands of insulin-stimulated muscle using a hyperinsulinemic-euglycemia clamp. We show that itgβ1 fl/fl SCLcre mice compared with itgβ1 fl/fl The microvasculature is an important site of resistance to muscle glucose uptake. We show that microvasculature integrins determine the exchange of glucose between the circulation and muscle. Specifically, a 30% reduction in the expression of endothelial integrin β1 subunit is sufficient to cause microcirculatory dysfunction and lead to insulin resistance. This emphasizes the importance of endothelial integrins in microcirculatory function and the importance of microcirculatory function for the ability of muscle to consume glucose.1 ) deficits in capillary flow rate, flow heterogeneity, and capillary density; 2 ) impaired insulin-stimulated glucose uptake despite sufficient transcapillary insulin efflux; and 3 ) reduced insulin-stimulated glucose uptake due to perfusion-limited glucose delivery. Thus, EC itgβ1 is necessary for microcirculatory function and to meet the metabolic challenge of insulin stimulation. NEW & NOTEWORTHY The microvasculature is an important site of resistance to muscle glucose uptake. We show that microvasculature integrins determine the exchange of glucose between the circulation and muscle. Specifically, a 30% reduction in the expression of endothelial integrin β1 subunit is sufficient to cause microcirculatory dysfunction and lead to insulin resistance. This emphasizes the importance of endothelial integrins in microcirculatory function and the importance of microcirculatory function for the ability of muscle to consume glucose.

Objective: Obesity increases deposition of extracellular matrix (ECM) components of cardiac tissue. Since obesity aggregates with insulin resistance and heart disease, it is imperative to determine whether the increased ECM deposition contributes to this disease cluster. The hypotheses tested in this study were that in cardiac tissue of obese mice i) increased deposition of ECM components (collagens and hyaluronan) contributes to cardiac insulin resistance and that a reduction in these components improves cardiac insulin action and ii) reducing excess collagens and hyaluronan mitigates obesity-associated cardiac dysfunction., Methods: Genetic and pharmacological approaches that manipulated collagen and hyaluronan contents were employed in obese C57BL/6 mice fed a high fat (HF) diet. Cardiac insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp and cardiac function was measured by pressure-volume loop analysis in vivo., Results: We demonstrated a tight association between increased ECM deposition with cardiac insulin resistance. Increased collagen deposition by genetic deletion of matrix metalloproteinase 9 (MMP9) exacerbated cardiac insulin resistance and pirfenidone, a clinically available anti-fibrotic medication which inhibits collagen expression, improved cardiac insulin resistance in obese mice. Furthermore, decreased hyaluronan deposition by treatment with PEGylated human recombinant hyaluronidase PH20 (PEGPH20) improved cardiac insulin resistance in obese mice. These relationships corresponded to functional changes in the heart. Both PEGPH20 and pirfenidone treatment in obese mice ameliorated HF diet-induced abnormal myocardial remodelling., Conclusion: Our results provide important new insights into the role of ECM deposition in the pathogenesis of cardiac insulin resistance and associated dysfunction in obesity of distinct mouse models. These findings support the novel therapeutic potential of targeting early cardiac ECM abnormalities in the prevention and treatment of obesity-related cardiovascular complications., Competing Interests: Declaration of competing interest The authors declare no conflicts of interests., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Mammals are protected from changes in environmental temperature by altering energetic processes that modify heat production. Insulin is the dominant stimulus of glucose uptake and metabolism, which are fundamental for thermogenic processes. The purpose of this work was to determine the interaction of ambient temperature induced changes in energy expenditure (EE) on the insulin sensitivity of glucose fluxes. Short-term and adaptive responses to thermoneutral temperature (TN, ∼28 °C) and room (laboratory) temperature (RT, ∼22 °C) were studied in mice. This range of temperature does not cause detectable changes in circulating catecholamines or shivering and postabsorptive glucose homeostasis is maintained. We tested the hypothesis that a decrease in EE that occurs with TN causes insulin resistance and that this reduction in insulin action and EE is reversed upon short term (<12h) transition to RT. Insulin-stimulated glucose disposal (Rd) and tissue-specific glucose metabolic index were assessed combining isotopic tracers with hyperinsulinemic-euglycemic clamps. EE and insulin-stimulated Rd are both decreased (∼50%) in TN-adapted vs RT-adapted mice. When RT-adapted mice are switched to TN, EE rapidly decreases and Rd is reduced by ∼50%. TN-adapted mice switched to RT exhibit a rapid increase in EE, but whole-body insulin-stimulated Rd remains at the low rates of TN-adapted mice. In contrast, whole body glycolytic flux rose with EE. This higher EE occurs without increasing glucose uptake from the blood, but rather by diverting glucose from glucose storage to glycolysis. In addition to adaptations in insulin action, 'insulin-independent' glucose uptake in brown fat is exquisitely sensitive to thermoregulation. These results show that insulin action adjusts to non-stressful changes in ambient temperature to contribute to the support of body temperature homeostasis without compromising glucose homeostasis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Obesity-associated complications are causing increasing morbidity and mortality worldwide. Expansion of adipose tissue in obesity leads to a state of low-grade chronic inflammation and dysregulated metabolism, resulting in insulin resistance and metabolic syndrome. Adipose tissue macrophages (ATMs) accumulate in obesity and are a source of proinflammatory cytokines that further aggravate adipocyte dysfunction. Macrophages are rich sources of cyclooxygenase (COX), the rate limiting enzyme for prostaglandin E2 (PGE2) production. When mice were fed a high-fat diet (HFD), ATMs increased expression of COX-2. Selective myeloid cell COX-2 deletion resulted in increased monocyte recruitment and proliferation of ATMs, leading to increased proinflammatory ATMs with decreased phagocytic ability. There were increased weight gain and adiposity, decreased peripheral insulin sensitivity and glucose utilization, increased adipose tissue inflammation and fibrosis, and abnormal adipose tissue angiogenesis. HFD pair-feeding led to similar increases in body weight, but mice with selective myeloid cell COX-2 still exhibited decreased peripheral insulin sensitivity and glucose utilization. Selective myeloid deletion of the macrophage PGE2 receptor subtype, EP4, produced a similar phenotype, and a selective EP4 agonist ameliorated the metabolic abnormalities seen with ATM COX-2 deletion. Therefore, these studies demonstrated that an ATM COX-2/PGE2/EP4 axis plays an important role in inhibiting adipose tissue dysfunction.

Objective: Increased deposition of the extracellular matrix (ECM) in adipose tissue (AT) during obesity contributes to insulin resistance. The integrin receptors transmit changes in the extracellular environment causing corresponding intracellular adaptations. Integrin-linked kinase (ILK), an adaptor protein, is a central hub for intracellular signaling of integrins. This study determined the role of ILK in adipose function and insulin resistance., Methods: The pathogenic role of ILK in obesity and insulin resistance was studied in human adipose tissue and adipocyte-specific ILK-deficient mice (ILK lox/lox AdCre). ILK lox/lox AdCre mice together with wild-type littermates (ILK lox/lox ) were fed a chow diet or 60% high-fat (HF) diet for 16 weeks. In vivo insulin sensitivity was determined by hyperinsulinemic-euglycemic clamps., Results: AT ILK expression was increased by HF diet feeding in mice and increased in visceral fat of morbidly obese humans. The HF-fed ILK lox/lox AdCre mice displayed reduced fat mass and improved glucose tolerance relative to the HF-fed ILK lox/lox mice. During a hyperinsulinemic-euglycemic clamp, the HF-fed ILK lox/lox AdCre mice exhibited partially improved insulin resistance in AT. Lipolysis was suppressed to a greater extent by insulin and glucose uptake in brown AT (BAT) increased. Increased inhibition of lipolysis may have been attributed to increased vascularization in white AT, while increased glucose uptake in BAT was associated with increased Akt phosphorylation and P38/JNK dephosphorylation. Notably, AT insulin sensitivity in lean mice was not affected by ILK deletion. Moreover, reduced fat mass in the HF-fed ILK lox/lox AdCre mice may have been attributed to decreased free fatty acid uptake into adipocytes via the downregulation of CD36 gene expression. Consistent with the results in the mice, knockdown and knockout of ILK in 3T3-L1 cells decreased lipid accumulation and CD36 gene expression during adipogenesis., Conclusions: These data show that adipocyte ILK is important for regulating HF diet-mediated insulin resistance in AT in a manner consistent with AT function., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Aberrant extracellular matrix (ECM) remodelling in muscle, liver and adipose tissue is a key characteristic of obesity and insulin resistance. Despite its emerging importance, the effective ECM targets remain largely undefined due to limitations of current approaches. Here, we developed a novel ECM-specific mass spectrometry-based proteomics technique to characterise the global view of the ECM changes in the skeletal muscle and liver of mice after high fat (HF) diet feeding. We identified distinct signatures of HF-induced protein changes between skeletal muscle and liver where the ECM remodelling was more prominent in the muscle than liver. In particular, most muscle collagen isoforms were increased by HF diet feeding whereas the liver collagens were differentially but moderately affected highlighting a different role of the ECM remodelling in different tissues of obesity. Moreover, we identified a novel association between collagen 24α1 and insulin resistance in the skeletal muscle. Using quantitative gene expression analysis, we extended this association to the white adipose tissue. Importantly, collagen 24α1 mRNA was increased in the visceral adipose tissue, but not the subcutaneous adipose tissue of obese diabetic subjects compared to lean controls, implying a potential pathogenic role of collagen 24α1 in obesity and type 2 diabetes.

Purpose: Diabetes mellitus is a delayed effect of radiation exposure in human and nonhuman primates. Diabetes mellitus is characterized by peripheral tissue insulin resistance, and as a result, irradiation exposure may cause important changes in insulin-sensitive tissues such as muscle and adipose., Methods and Materials: We prospectively investigated changes in response to irradiation (4 Gy whole body exposure) in 16 male rhesus macaques. We evaluated changes in body composition and glycemic control for 2 years. Insulin responsiveness, lipolysis, inflammation, and fibrosis were evaluated at study end., Results: Irradiated animals accumulate less fat and significantly increased percent glycation of hemoglobin A1c over time, such that 40% of irradiated monkeys had values that define them as diabetic at 2 years. Subcutaneous (SQ) adipose tissue was insulin resistant, as evidenced by reduced phosphorylation of the insulin receptor substrate-1 in response to insulin challenge and had increased basal lipolysis despite comparable insulin exposures to control animals. Irradiated SQ adipose tissue had more macrophage infiltration and adipocytes were larger. The observed hypertrophy was associated with decreased glycemic control and macrophage infiltration correlated with decreased adiponectin, signifying that inflammation is associated with worsening health. No evidence of SQ adipose fibrosis was detected., Conclusions: Our study is the first to prospectively illustrate that sublethal irradiation exposures directly propagate metabolic disease in the absence of obesity in nonhuman primates and implicate SQ adipose dysfunction as a target tissue., (Copyright © 2019 Elsevier Inc. All rights reserved.)

This study sought to examine the functional significance of mitochondrial protein acetylation using a double knockout (DKO) mouse model harboring muscle-specific deficits in acetyl-CoA buffering and lysine deacetylation, due to genetic ablation of carnitine acetyltransferase and Sirtuin 3, respectively. DKO mice are highly susceptible to extreme hyperacetylation of the mitochondrial proteome and develop a more severe form of diet-induced insulin resistance than either single KO mouse line. However, the functional phenotype of hyperacetylated DKO mitochondria is largely normal. Of the >120 measures of respiratory function assayed, the most consistently observed traits of a markedly heightened acetyl-lysine landscape are enhanced oxygen flux in the context of fatty acid fuel and elevated rates of electron leak. In sum, the findings challenge the notion that lysine acetylation causes broad-ranging damage to mitochondrial quality and performance and raise the possibility that acetyl-lysine turnover, rather than acetyl-lysine stoichiometry, modulates redox balance and carbon flux., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Extracellular matrix hyaluronan is increased in skeletal muscle of high-fat-fed insulin-resistant mice, and reduction of hyaluronan by PEGPH20 hyaluronidase ameliorates diet-induced insulin resistance (IR). CD44, the main hyaluronan receptor, is positively correlated with type 2 diabetes. This study determines the role of CD44 in skeletal muscle IR. Global CD44-deficient ( cd44 -/- ) mice and wild-type littermates ( cd44 +/+ ) were fed a chow diet or 60% high-fat diet for 16 wk. High-fat-fed cd44 -/- mice were also treated with PEGPH20 to evaluate its CD44-dependent action. Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp (ICv). High-fat feeding increased muscle CD44 protein expression. In the absence of differences in body weight and composition, despite lower clamp insulin during ICv, the cd44 -/- mice had sustained glucose infusion rate (GIR) regardless of diet. High-fat diet-induced muscle IR as evidenced by decreased muscle glucose uptake (Rg) was exhibited in cd44 +/+ mice but absent in cd44 -/- mice. Moreover, gastrocnemius Rg remained unchanged between genotypes on chow diet but was increased in high-fat-fed cd44 -/- compared with cd44 +/+ when normalized to clamp insulin concentrations. Ameliorated muscle IR in high-fat-fed cd44 -/- mice was associated with increased vascularization. In contrast to previously observed increases in wild-type mice, PEGPH20 treatment in high-fat-fed cd44 -/- mice did not change GIR or muscle Rg during ICv, suggesting a CD44-dependent action. In conclusion, genetic CD44 deletion improves muscle IR, and the beneficial effects of PEGPH20 are CD44-dependent. These results suggest a critical role of CD44 in promoting hyaluronan-mediated muscle IR, therefore representing a potential therapeutic target for diabetes.

Sepsis costs the healthcare system $23 billion annually and has a mortality rate between 10 and 40%. An early indication of sepsis is the onset of hyperglycemia, which is the result of sepsis-induced insulin resistance in skeletal muscle. Previous investigations have focused on events in the myocyte (e.g., insulin signaling and glucose transport and subsequent metabolism) as the causes for this insulin-resistant state. However, the delivery of insulin to the skeletal muscle is also an important determinant of insulin action. Skeletal muscle microvascular blood flow, which delivers the insulin to the muscle, is known to be decreased during sepsis. Here we test whether the reduced capillary blood flow to skeletal muscle belies the sepsis-induced insulin resistance by reducing insulin delivery to the myocyte. We hypothesize that decreased capillary flow and consequent decrease in insulin delivery is an early event that precedes gross cardiovascular alterations seen with sepsis. This hypothesis was examined in mice treated with either lipopolysaccharide (LPS) or polymicrobial sepsis followed by intravital microscopy of the skeletal muscle microcirculation. We calculated insulin delivery to the myocyte using two independent methods and found that LPS and sepsis rapidly reduce insulin delivery to the skeletal muscle by ~50%; this was driven by decreases in capillary flow velocity and the number of perfused capillaries. Furthermore, the changes in skeletal muscle microcirculation occur before changes in both cardiac output and arterial blood pressure. These data suggest that a rapid reduction in skeletal muscle insulin delivery contributes to the induction of insulin resistance during sepsis.

Integrin-linked kinase (ILK) is a critical intracellular signaling node for integrin receptors. Its role in liver development is complex, as ILK deletion at E10.5 (before hepatocyte differentiation) results in biochemical and morphological differences that resolve as mice age. Nevertheless, mice with ILK depleted specifically in hepatocytes are protected from the hepatic insulin resistance during obesity. Despite the potential importance of hepatocyte ILK to metabolic health, it is unknown how ILK controls hepatic metabolism or glucoregulation. The present study tested the role of ILK in hepatic metabolism and glucoregulation by deleting it specifically in hepatocytes, using a cre-lox system that begins expression at E15.5 (after initiation of hepatocyte differentiation). These mice develop the most severe morphological and glucoregulatory abnormalities at 6 wk, but these gradually resolve with age. After identifying when the deletion of ILK caused a severe metabolic phenotype, in depth studies were performed at this time point to define the metabolic programs that coordinate control of glucoregulation that are regulated by ILK. We show that 6-wk-old ILK-deficient mice have higher glucose tolerance and decreased net glycogen synthesis. Additionally, ILK was shown to be necessary for transcription of mitochondrial-related genes, oxidative metabolism, and maintenance of cellular energy status. Thus, ILK is required for maintaining hepatic transcriptional and metabolic programs that sustain oxidative metabolism, which are required for hepatic maintenance of glucose homeostasis.

The NAD+-dependent deacetylase SIRT2 is unique amongst sirtuins as it is effective in the cytosol, as well as the mitochondria. Defining the role of cytosolic acetylation state in specific tissues is difficult since even physiological effects at the whole body level are unknown. We hypothesized that genetic SIRT2 knockout (KO) would lead to impaired insulin action, and that this impairment would be worsened in HF fed mice. Insulin sensitivity was tested using the hyperinsulinemic-euglycemic clamp in SIRT2 KO mice and WT littermates. SIRT2 KO mice exhibited reduced skeletal muscle insulin-induced glucose uptake compared to lean WT mice, and this impairment was exacerbated in HF SIRT2 KO mice. Liver insulin sensitivity was unaffected in lean SIRT2 KO mice. However, the insulin resistance that accompanies HF-feeding was worsened in SIRT2 KO mice. It was notable that the effects of SIRT2 KO were largely disassociated from cytosolic acetylation state, but were closely linked to acetylation state in the mitochondria. SIRT2 KO led to an increase in body weight that was due to increased food intake in HF fed mice. In summary, SIRT2 deletion in vivo reduces muscle insulin sensitivity and contributes to liver insulin resistance by a mechanism that is unrelated to cytosolic acetylation state. Mitochondrial acetylation state and changes in feeding behavior that result in increased body weight correspond to the deleterious effects of SIRT2 KO on insulin action., Competing Interests: The authors have declared that no competing interests exist.

In clinical trials, inhibition of cholesteryl ester transfer protein (CETP) raises HDL cholesterol levels but does not robustly improve cardiovascular outcomes. Approximately two-thirds of trial participants are obese. Lower plasma CETP activity is associated with increased cardiovascular risk in human studies, and protective aspects of CETP have been observed in mice fed a high-fat diet (HFD) with regard to metabolic outcomes. To define whether CETP inhibition has different effects depending on the presence of obesity, we performed short-term anacetrapib treatment in chow- and HFD-fed CETP transgenic mice. Anacetrapib raised HDL cholesterol and improved aspects of HDL functionality, including reverse cholesterol transport, and HDL's antioxidative capacity in HFD-fed mice was better than in chow-fed mice. Anacetrapib worsened the anti-inflammatory capacity of HDL in HFD-fed mice. The HDL proteome was markedly different with anacetrapib treatment in HFD- versus chow-fed mice. Despite benefits on HDL, anacetrapib led to liver triglyceride accumulation and insulin resistance in HFD-fed mice. Overall, our results support a physiologic importance of CETP in protecting from fatty liver and demonstrate context selectivity of CETP inhibition that might be important in obese subjects., (© 2018 by the American Diabetes Association.)

The liver extracellular matrix (ECM) expands with high-fat (HF) feeding. This finding led us to address whether receptors for the ECM, integrins, are key to the development of diet-induced hepatic insulin resistance. Integrin-linked kinase (ILK) is a downstream integrin signaling molecule involved in multiple hepatic processes, including those related to differentiation, wound healing, and metabolism. We tested the hypothesis that deletion of ILK in mice on an HF diet would disrupt the ECM-integrin signaling axis, thereby preventing the transformation into the insulin-resistant liver. To determine the role of ILK in hepatic insulin action in vivo, male C57BL/6J ILK lox/lox mice were crossed with Albcre mice to produce a hepatocyte-specific ILK deletion (ILK lox/lox Albcre). Results from this study show that hepatic ILK deletion has no effect on insulin action in lean mice but sensitizes the liver to insulin during the challenge of HF feeding. This effect corresponds to changes in the expression and activation of key insulin signaling pathways as well as a greater capacity for hepatic mitochondrial glucose oxidation. This demonstrates that ILK contributes to hepatic insulin resistance and highlights the previously undefined role of integrin signaling in the pathogenesis of diet-induced hepatic insulin resistance., (© 2017 by the American Diabetes Association.)

Diet-induced muscle insulin resistance is associated with expansion of extracellular matrix (ECM) components, such as collagens, and the expression of collagen-binding integrin, α2β1. Integrins transduce signals from ECM via their cytoplasmic domains, which bind to intracellular integrin-binding proteins. The integrin-linked kinase (ILK)-PINCH-parvin (IPP) complex interacts with the cytoplasmic domain of β-integrin subunits and is critical for integrin signaling. In this study we defined the role of ILK, a key component of the IPP complex, in diet-induced muscle insulin resistance. Wild-type (ILK(lox/lox)) and muscle-specific ILK-deficient (ILK(lox/lox)HSAcre) mice were fed chow or a high-fat (HF) diet for 16 weeks. Body weight was not different between ILK(lox/lox) and ILK(lox/lox)HSAcre mice. However, HF-fed ILK(lox/lox)HSAcre mice had improved muscle insulin sensitivity relative to HF-fed ILK(lox/lox) mice, as shown by increased rates of glucose infusion, glucose disappearance, and muscle glucose uptake during a hyperinsulinemic-euglycemic clamp. Improved muscle insulin action in the HF-fed ILK(lox/lox)HSAcre mice was associated with increased insulin-stimulated phosphorylation of Akt and increased muscle capillarization. These results suggest that ILK expression in muscle is a critical component of diet-induced insulin resistance, which possibly acts by impairing insulin signaling and insulin perfusion through capillaries., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Angiotensin-(1-7) improves glycemic control in animal models of cardiometabolic syndrome. The tissue-specific sites of action and blood pressure dependence of these metabolic effects, however, remain unclear. We hypothesized that Ang-(1-7) improves insulin sensitivity by enhancing peripheral glucose delivery. Adult male C57BL/6J mice were placed on standard chow or 60% high-fat diet for 11 weeks. Ang-(1-7) (400 ng/kg per minute) or saline was infused subcutaneously during the last 3 weeks of diet, and hyperinsulinemic-euglycemic clamps were performed at the end of treatment. High-fat fed mice exhibited modest hypertension (systolic blood pressure: 137 ± 3 high fat versus 123 ± 5 mm Hg chow;P=0.001), which was not altered by Ang-(1-7) (141 ± 4 mm Hg;P=0.574). Ang-(1-7) did not alter body weight or fasting glucose and insulin in chow or high-fat fed mice. Ang-(1-7) increased the steady-state glucose infusion rate needed to maintain euglycemia in high-fat fed mice (31 ± 5 Ang-(1-7) versus 16 ± 1 mg/kg per minute vehicle;P=0.017) reflecting increased whole-body insulin sensitivity, with no effect in chow-fed mice. The improved insulin sensitivity in high-fat fed mice was because of an enhanced rate of glucose disappearance (34 ± 5 Ang-(1-7) versus 20 ± 2 mg/kg per minute vehicle;P=0.049). Ang-(1-7) enhanced glucose uptake specifically into skeletal muscle by increasing translocation of glucose transporter 4 to the sarcolemma. Our data suggest that Ang-(1-7) has direct insulin-sensitizing effects on skeletal muscle, independent of changes in blood pressure. These findings provide new insight into mechanisms by which Ang-(1-7) improves insulin action, and provide further support for targeting this peptide in cardiometabolic disease., (© 2016 American Heart Association, Inc.)

Protein hyperacetylation is associated with glucose intolerance and insulin resistance, suggesting that the enzymes regulating the acetylome play a role in this pathological process. Sirtuin 3 (SIRT3), the primary mitochondrial deacetylase, has been linked to energy homeostasis. Thus, it is hypothesized that the dysregulation of the mitochondrial acetylation state, via genetic deletion of SIRT3, will amplify the deleterious effects of a high-fat diet (HFD). Hyperinsulinemic-euglycemic clamp experiments show, for the first time, that mice lacking SIRT3 exhibit increased insulin resistance due to defects in skeletal muscle glucose uptake. Permeabilized muscle fibers from HFD-fed SIRT3 knockout (KO) mice showed that tricarboxylic acid cycle substrate-based respiration is decreased while fatty acid-based respiration is increased, reflecting a fuel switch from glucose to fatty acids. Consistent with reduced muscle glucose uptake, hexokinase II (HKII) binding to the mitochondria is decreased in muscle from HFD-fed SIRT3 KO mice, suggesting decreased HKII activity. These results show that the absence of SIRT3 in HFD-fed mice causes profound impairments in insulin-stimulated muscle glucose uptake, creating an increased reliance on fatty acids. Insulin action was not impaired in the lean SIRT3 KO mice. This suggests that SIRT3 protects against dietary insulin resistance by facilitating glucose disposal and mitochondrial function., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

The extracellular matrix (ECM) is a highly-dynamic compartment that undergoes remodeling as a result of injury and repair. Over the past decade, mounting evidence in humans and rodents suggests that ECM remodeling is associated with diet-induced insulin resistance in several metabolic tissues. In addition, integrin receptors for the ECM have also been implicated in the regulation of insulin action. This review addresses what is currently known about the ECM, integrins, and insulin action in the muscle, liver, and adipose tissue. Understanding how ECM remodeling and integrin signaling regulate insulin action may aid in the development of new therapeutic targets for the treatment of insulin resistance and type 2 diabetes (T2D)., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Hepatic insulin resistance is associated with increased collagen. Integrin α1β1 is a collagen-binding receptor expressed on hepatocytes. Here, we show that expression of the α1 subunit is increased in hepatocytes isolated from high fat (HF)-fed mice. To determine whether the integrin α1 subunit protects against impairments in hepatic glucose metabolism, we analyzed glucose tolerance and insulin sensitivity in HF-fed integrin α1-null (itga1(-/-)) and wild-type (itga1(+/+)) littermates. Using the insulin clamp, we found that insulin-stimulated hepatic glucose production was suppressed by ∼50% in HF-fed itga1(+/+) mice. In contrast, it was not suppressed in HF-fed itga1(-/-) mice, indicating severe hepatic insulin resistance. This was associated with decreased hepatic insulin signaling in HF-fed itga1(-/-) mice. Interestingly, hepatic triglyceride and diglyceride contents were normalized to chow-fed levels in HF-fed itga1(-/-) mice. This indicates that hepatic steatosis is dissociated from insulin resistance in HF-fed itga1(-/-) mice. The decrease in hepatic lipid accumulation in HF-fed itga1(-/-) mice was associated with altered free fatty acid metabolism. These studies establish a role for integrin signaling in facilitating hepatic insulin action while promoting lipid accumulation in mice challenged with a HF diet., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Aims/hypothesis: Increased extracellular matrix (ECM) collagen is a characteristic of muscle insulin resistance. Matrix metalloproteinase (MMP) 9 is a primary enzyme that degrades collagen IV (ColIV). As a component of the basement membrane, ColIV plays a key role in ECM remodelling. We tested the hypotheses that genetic deletion of MMP9 in mice increases muscle ColIV, induces insulin resistance in lean mice and worsens diet-induced muscle insulin resistance., Methods: Wild-type (Mmp9(+/+)) and Mmp9-null (Mmp9(-/-)) mice were chow or high-fat (HF) fed for 16 weeks. Insulin action was measured by the hyperinsulinaemic-euglycaemic clamp in conscious weight-matched surgically catheterised mice., Results: Mmp9(-/-) and HF feeding independently increased muscle ColIV. ColIV in HF-fed Mmp9(-/-) mice was further increased. Mmp9(-/-) did not affect fasting insulin or glucose in chow- or HF-fed mice. The glucose infusion rate (GIR), endogenous glucose appearance (EndoRa) and glucose disappearance (Rd) rates, and a muscle glucose metabolic index (Rg), were the same in chow-fed Mmp9(+/+) and Mmp9(-/-) mice. In contrast, HF-fed Mmp9(-/-) mice had decreased GIR, insulin-stimulated increase in Rd and muscle Rg. Insulin-stimulated suppression of EndoRa, however, remained the same in HF-fed Mmp9(-/-) and Mmp9(+/+) mice. Decreased muscle Rg in HF-fed Mmp9(-/-) was associated with decreased muscle capillaries., Conclusions/interpretation: Despite increased muscle ColIV, genetic deletion of MMP9 does not induce insulin resistance in lean mice. In contrast, this deletion results in a more profound state of insulin resistance, specifically in the skeletal muscle of HF-fed mice. These results highlight the importance of ECM remodelling in determining muscle insulin resistance in the presence of HF diet.

Background: This study aimed to evaluate the efficacy of mesenchymal stem cell (MSC) transplantation to mitigate abnormalities in cardiac-specific and systemic metabolism mediated by a combination of a myocardial infarction and diet-induced insulin resistance., Methods: C57BL/6 mice were high-fat fed for eight weeks prior to induction of a myocardial infarction via chronic ligation of the left anterior descending coronary artery. MSCs were administered directly after myocardial infarction induction through a single intramyocardial injection. Echocardiography was performed prior to the myocardial infarction as well as seven and 28 days post-myocardial infarction. Hyperinsulinemic-euglycemic clamps coupled with 2-[14C]deoxyglucose were employed 36 days post-myocardial infarction (13 weeks of high-fat feeding) to assess systemic insulin sensitivity and insulin-mediated, tissue-specific glucose uptake in the conscious, unrestrained mouse. High-resolution respirometry was utilized to evaluate cardiac mitochondrial function in saponin-permeabilized cardiac fibers., Results: MSC administration minimized the decline in ejection fraction following the myocardial infarction. The greater systolic function in MSC-treated mice was associated with increased in vivo cardiac glucose uptake and enhanced mitochondrial oxidative phosphorylation efficiency. MSC therapy promoted reductions in fasting arterial glucose and fatty acid concentrations. Additionally, glucose uptake in peripheral tissues including skeletal muscle and adipose tissue was elevated in MSC-treated mice. Enhanced glucose uptake in these tissues was associated with improved insulin signalling as assessed by Akt phosphorylation and prevention of a decline in GLUT4 often associated with high-fat feeding., Conclusions: These studies provide insight into the utility of MSC transplantation as a metabolic therapy that extends beyond the heart exerting beneficial systemic effects on insulin action.

The endogenous hormone relaxin increases vascular reactivity and angiogenesis. We demonstrate that acute relaxin infusion in lean C57BL/6J mice enhances skeletal muscle perfusion and augments muscle glucose uptake during a hyperinsulinemic-euglycemic clamp. However, an acute effect was absent in mice fed a high-fat (HF) diet for 13 weeks. In contrast, mice fed an HF diet for 13 weeks and continuously treated with relaxin for the final 3 weeks of the diet exhibited decreased fasting blood glucose. Insulin-stimulated whole-body glucose disappearance and percent suppression of hepatic glucose production are corrected by chronic relaxin. The increase in peripheral glucose utilization is a result of augmented in vivo skeletal muscle glucose uptake. Relaxin intervention improves endothelial-dependent vascular reactivity and induces a two-fold proliferation in skeletal muscle capillarity. The metabolic effects of the treatment are not attributed to changes in myocellular insulin signaling. Relaxin intervention reverses the accumulation of collagen III in the liver and collagen III and collagen IV in the heart; this is induced by HF feeding. These studies show the potential of relaxin in the treatment of diet-induced insulin resistance and vascular dysfunction. Relaxin provides a novel therapeutic approach targeting the extramyocellular barriers to insulin action, which are critical to the pathogenesis of insulin resistance.

Increased deposition of specific extracellular matrix (ECM) components is a characteristic of insulin-resistant skeletal muscle. Hyaluronan (HA) is a major constituent of the ECM. The hypotheses that 1) HA content is increased in the ECM of insulin-resistant skeletal muscle and 2) reduction of HA in the muscle ECM by long-acting pegylated human recombinant PH20 hyaluronidase (PEGPH20) reverses high-fat (HF) diet-induced muscle insulin resistance were tested. We show that muscle HA was increased in HF diet-induced obese (DIO) mice and that treatment of PEGPH20, which dose-dependently reduced HA in muscle ECM, decreased fat mass, adipocyte size, and hepatic and muscle insulin resistance in DIO mice at 10 mg/kg. Reduced muscle insulin resistance was associated with increased insulin signaling, muscle vascularization, and percent cardiac output to muscle rather than insulin sensitization of muscle per se. Dose-response studies revealed that PEGPH20 dose-dependently increased insulin sensitivity in DIO mice with a minimally effective dose of 0.01 mg/kg. PEGPH20 at doses of 0.1 and 1 mg/kg reduced muscle HA to levels seen in chow-fed mice, decreased fat mass, and increased muscle glucose uptake. These findings suggest that ECM HA is a target for treatment of insulin resistance.

Background: Disruption of circadian (daily) timekeeping enhances the risk of metabolic syndrome, obesity, and type 2 diabetes. While clinical observations have suggested that insulin action is not constant throughout the 24 hr cycle, its magnitude and periodicity have not been assessed. Moreover, when circadian rhythmicity is absent or severely disrupted, it is not known whether insulin action will lock to the peak, nadir, or mean of the normal periodicity of insulin action., Results: We used hyperinsulinemic-euglycemic clamps to show a bona fide circadian rhythm of insulin action; mice are most resistant to insulin during their daily phase of relative inactivity. Moreover, clock-disrupted Bmal1-knockout mice are locked into the trough of insulin action and lack rhythmicity in insulin action and activity patterns. When rhythmicity is rescued in the Bmal1-knockout mice by expression of the paralogous gene Bmal2, insulin action and activity patterns are restored. When challenged with a high-fat diet, arhythmic mice (either Bmal1-knockout mice or wild-type mice made arhythmic by exposure to constant light) were obese prone. Adipose tissue explants obtained from high-fat-fed mice have their own periodicity that was longer than animals on a chow diet., Conclusions: This study provides rigorous documentation for a circadian rhythm of insulin action and demonstrates that disturbing the natural rhythmicity of insulin action will disrupt the rhythmic internal environment of insulin sensitive tissue, thereby predisposing the animals to insulin resistance and obesity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Muscle insulin resistance is associated with a reduction in vascular endothelial growth factor (VEGF) action and muscle capillary density. We tested the hypothesis that muscle capillary rarefaction critically contributes to the etiology of muscle insulin resistance in chow-fed mice with skeletal and cardiac muscle VEGF deletion (mVEGF(-/-)) and wild-type littermates (mVEGF(+/+)) on a C57BL/6 background. The mVEGF(-/-) mice had an ~60% and ~50% decrease in capillaries in skeletal and cardiac muscle, respectively. The mVEGF(-/-) mice had augmented fasting glucose turnover. Insulin-stimulated whole-body glucose disappearance was blunted in mVEGF(-/-) mice. The reduced peripheral glucose utilization during insulin stimulation was due to diminished in vivo cardiac and skeletal muscle insulin action and signaling. The decreased insulin-stimulated muscle glucose uptake was independent of defects in insulin action at the myocyte, suggesting that the impairment in insulin-stimulated muscle glucose uptake was due to poor muscle perfusion. The deletion of VEGF in cardiac muscle did not affect cardiac output. These studies emphasize the importance for novel therapeutic approaches that target the vasculature in the treatment of insulin-resistant muscle.

AS160 (Akt substrate of 160 kDa) is a Rab GTPase-activating protein implicated in insulin control of GLUT4 (glucose transporter 4) trafficking. In humans, a truncation mutation (R363X) in one allele of AS160 decreased the expression of the protein and caused severe postprandial hyperinsulinaemia during puberty. To complement the limited studies possible in humans, we generated an AS160-knockout mouse. In wild-type mice, AS160 expression is relatively high in adipose tissue and soleus muscle, low in EDL (extensor digitorum longus) muscle and detectable in liver only after enrichment. Despite having lower blood glucose levels under both fasted and random-fed conditions, the AS160-knockout mice exhibited insulin resistance in both muscle and liver in a euglycaemic clamp study. Consistent with this paradoxical phenotype, basal glucose uptake was higher in AS160-knockout primary adipocytes and normal in isolated soleus muscle, but their insulin-stimulated glucose uptake and overall GLUT4 levels were markedly decreased. In contrast, insulin-stimulated glucose uptake and GLUT4 levels were normal in EDL muscle. The liver also contributes to the AS160-knockout phenotype via hepatic insulin resistance, elevated hepatic expression of phosphoenolpyruvate carboxykinase isoforms and pyruvate intolerance, which are indicative of increased gluconeogenesis. Overall, as well as its catalytic function, AS160 influences expression of other proteins, and its loss deregulates basal and insulin-regulated glucose homoeostasis, not only in tissues that normally express AS160, but also by influencing liver function.

Obesity triggers a low-grade systemic inflammation, which plays an important role in the development of obesity-associated metabolic diseases. In searching for links between lipid accumulation and chronic inflammation, we examined invariant natural killer T (iNKT) cells, a subset of T lymphocytes that react with lipids and regulate inflammatory responses. We show that iNKT cells respond to dietary lipid excess and become activated before or at the time of tissue recruitment of inflammatory leukocytes, and that these cells progressively increase proinflammatory cytokine production in obese mice. Such iNKT cells skew other leukocytes toward proinflammatory cytokine production and induce an imbalanced proinflammatory cytokine environment in multiple tissues. Further, iNKT cell deficiency ameliorates tissue inflammation and provides protection against obesity-induced insulin resistance and hepatic steatosis. Conversely, chronic iNKT cell stimulation using a canonical iNKT cell agonist exacerbates tissue inflammation and obesity-associated metabolic disease. These findings place iNKT cells into the complex network linking lipid excess to inflammation in obesity and suggest new therapeutic avenues for obesity-associated metabolic disorders.

Although obesity is associated with overactivation of the white adipose tissue (WAT) renin-angiotensin system (RAS), a causal link between the latter and systemic insulin resistance is not established. We tested the hypothesis that overexpression of angiotensinogen (Agt) from WAT causes systemic insulin resistance via modulation of adipose inflammation. Glucose tolerance, systemic insulin sensitivity, and WAT inflammatory markers were analyzed in mice overexpressing Agt in the WAT (aP2-Agt mice). Proteomic studies and in vitro studies using 3T3-L1 adipocytes were performed to build a mechanistic framework. Male aP2-Agt mice exhibited glucose intolerance, insulin resistance, and lower insulin-stimulated glucose uptake by the skeletal muscle. The difference in glucose tolerance between genotypes was normalized by high-fat (HF) feeding, and was significantly improved by treatment with angiotensin-converting enzyme (ACE) inhibitor captopril. aP2-Agt mice also had higher monocyte chemotactic protein-1 (MCP-1) and lower interleukin-10 (IL-10) in the WAT, indicating adipose inflammation. Proteomic studies in WAT showed that they also had higher monoglyceride lipase (MGL) and glycerol-3-phosphate dehydrogenase levels. Treatment with angiotensin II (Ang II) increased MCP-1 and resistin secretion from adipocytes, which was prevented by cotreating with inhibitors of the nuclear factor-κB (NF-κB) pathway or nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In conclusion, we show for the first time that adipose RAS overactivation causes glucose intolerance and systemic insulin resistance. The mechanisms appear to be via reduced skeletal muscle glucose uptake, at least in part due to Ang II-induced, NADPH oxidase and NFκB-dependent increases in WAT inflammation.

Objective: To assess the effects of different bariatric surgical procedures on the treatment of obesity and insulin resistance in high fat diet-induced obese (DIO) mice., Background: Bariatric surgery is currently considered the most effective treatment for morbid obesity and its comorbidities; however, a systematic study of their mechanisms is still lacking., Methods: We developed bariatric surgery models, including gastric banding, sleeve gastrectomy, Roux-en-Y gastric bypass (RYGB), modified RYGB (mRYGB) and biliopancreatic diversion (BPD), in DIO mice. Body weight, body fat and lean mass, liver steatosis, glucose tolerance and pancreatic beta cell function were examined., Results: All bariatric surgeries resulted in significant weight loss, reduced body fat and improved glucose tolerance in the short term (4 weeks), compared with mice with sham surgery. Of the bariatric surgery models, sleeve gastrectomy and mRYGB had higher success rates and lower mortalities and represent reliable restrictive and gastrointestinal (GI) bypass mouse bariatric surgery models, respectively. In the long term, the GI bypass procedure produced more profound weight loss, significant improvement of glucose tolerance and liver steatosis than the restrictive procedure. DIO mice had increased insulin promoter activity, suggesting overactivation of pancreatic beta cells, which was regulated by the mRYGB procedure. Compared with the restrictive procedure, the GI bypass procedure showed more severe symptoms of malnutrition following bariatric surgery., Discussions: Both restrictive and GI bypass procedures provide positive effects on weight loss, fat composition, liver steatosis and glucose tolerance; however, in the long term, the GI bypass shows better results than restrictive procedures.

Objective: The hypothesis that high-fat (HF) feeding causes skeletal muscle extracellular matrix (ECM) remodeling in C57BL/6J mice and that this remodeling contributes to diet-induced muscle insulin resistance (IR) through the collagen receptor integrin α(2)β(1) was tested., Research Design and Methods: The association between IR and ECM remodeling was studied in mice fed chow or HF diet. Specific genetic and pharmacological murine models were used to study effects of HF feeding on ECM in the absence of IR. The role of ECM-integrin interaction in IR was studied using hyperinsulinemic-euglycemic clamps on integrin α(2)β(1)-null (itga2(-/-)), integrin α(1)β(1)-null (itga1(-/-)), and wild-type littermate mice fed chow or HF. Integrin α(2)β(1) and integrin α(1)β(1) signaling pathways have opposing actions., Results: HF-fed mice had IR and increased muscle collagen (Col) III and ColIV protein; the former was associated with increased transcript, whereas the latter was associated with reduced matrix metalloproteinase 9 activity. Rescue of muscle IR by genetic muscle-specific mitochondria-targeted catalase overexpression or by the phosphodiesterase 5a inhibitor, sildenafil, reversed HF feeding effects on ECM remodeling and increased muscle vascularity. Collagen remained elevated in HF-fed itga2(-/-) mice. Nevertheless, muscle insulin action and vascularity were increased. Muscle IR in HF-fed itga1(-/-) mice was unchanged. Insulin sensitivity in chow-fed itga1(-/-) and itga2(-/-) mice was not different from wild-type littermates., Conclusions: ECM collagen expansion is tightly associated with muscle IR. Studies with itga2(-/-) mice provide mechanistic insight for this association by showing that the link between muscle IR and increased collagen can be uncoupled by the absence of collagen-integrin α(2)β(1) interaction.

Glucagon-like peptide-1 augments nutrient-stimulated insulin secretion. Chow-fed mice lacking the glucagon-like peptide-1 receptor (Glp1r) exhibit enhanced insulin-stimulated muscle glucose uptake but impaired suppression of endogenous glucose appearance (endoRa). This proposes a novel role for the Glp1r to regulate the balance of glucose disposal in muscle and liver by modulating insulin action. Whether this is maintained in an insulin-resistant state is unknown. The present studies tested the hypothesis that disruption of Glp1r expression overcomes high-fat (HF) diet-induced muscle insulin resistance and exacerbates HF diet-induced hepatic insulin resistance. Mice with a functional disruption of the Glp1r (Glp1r-/-) were compared with wild-type littermates (Glp1r+/+) after 12 wk on a regular chow diet or a HF diet. Arterial and venous catheters were implanted for sampling and infusions. Hyperinsulinemic-euglycemic clamps were performed on weight-matched male mice. [3-(3)H]glucose was used to determine glucose turnover, and 2[14C]deoxyglucose was used to measure the glucose metabolic index, an indicator of glucose uptake. Glp1r-/- mice exhibited increased glucose disappearance and muscle glucose metabolic index on either diet. This was associated with enhanced activation of muscle Akt and AMP-activated protein kinase and reduced muscle triglycerides in HF-fed Glp1r-/- mice. Chow-fed Glp1r-/- mice exhibited impaired suppression of endoRa and hepatic insulin signaling. In contrast, HF-fed Glp1r-/- mice exhibited improved suppression of endoRa and hepatic Akt activation. This was associated with decreased hepatic triglycerides and impaired activation of sterol regulatory element-binding protein-1. These results show that mice lacking the Glp1r are protected from HF diet-induced muscle and hepatic insulin resistance independent of effects on total fat mass.

Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator shown to improve glycemic control. However, the molecular and functional mechanisms underlying FGF21-mediated improvements in glycemic control are not completely understood. We examined FGF21 effects on insulin sensitivity and glucose fluxes upon chronic (daily injection for 8 d) and acute (6 h infusion) administration in ob/+ and ob/ob mice. Results show that chronic FGF21 ameliorated fasting hyperglycemia in ob/ob mice via increased glucose disposal and improved hepatic insulin sensitivity. Acute FGF21 suppressed hepatic glucose production, increased liver glycogen, lowered glucagon, and improved glucose clearance in ob/+ mice. These effects were blunted in ob/ob mice. Neither chronic nor acute FGF21 altered skeletal muscle or adipose tissue glucose uptake in either genotype. In conclusion, FGF21 has potent glycemic effects caused by hepatic changes in glucose flux and improved insulin sensitivity. Thus, these studies define mechanisms underlying anti-hyperglycemic actions of FGF21 and support its therapeutic potential.

High dietary fat intake leads to insulin resistance in skeletal muscle, and this represents a major risk factor for type 2 diabetes and cardiovascular disease. Mitochondrial dysfunction and oxidative stress have been implicated in the disease process, but the underlying mechanisms are still unknown. Here we show that in skeletal muscle of both rodents and humans, a diet high in fat increases the H(2)O(2)-emitting potential of mitochondria, shifts the cellular redox environment to a more oxidized state, and decreases the redox-buffering capacity in the absence of any change in mitochondrial respiratory function. Furthermore, we show that attenuating mitochondrial H(2)O(2) emission, either by treating rats with a mitochondrial-targeted antioxidant or by genetically engineering the overexpression of catalase in mitochondria of muscle in mice, completely preserves insulin sensitivity despite a high-fat diet. These findings place the etiology of insulin resistance in the context of mitochondrial bioenergetics by demonstrating that mitochondrial H(2)O(2) emission serves as both a gauge of energy balance and a regulator of cellular redox environment, linking intracellular metabolic balance to the control of insulin sensitivity.

Insulin resistance is characterized by elevated rates of cardiac fatty acid utilization resulting in reduced efficiency and cardiomyopathy. One potential therapeutic approach is to limit the uptake and oxidation of fatty acids. The aims of this study were to determine whether a quantitative reduction in heart-type fatty acid binding protein (FABP3) normalizes cardiac substrate utilization without altering cardiac function. Transgenic (FABP3(+/-)) and wild-type (WT) littermates were studied following low fat (LF) or high fat (HF) diets, with HF resulting in obese, insulin-resistant mice. Cardiovascular function (systolic blood pressure, % fractional shortening) and heart dimension were measured at weaning and every month afterward for 3 mo. During this period cardiovascular function was the same independent of genotype and diet. Catheters were surgically implanted in the carotid artery and jugular vein for sampling and infusions in mice at 4 mo of age. Following 5 d recovery, mice underwent either a saline infusion or a hyperinsulinemic-euglycemic clamp (4 mU kg(-1) min(-1)). Indices of long chain fatty acid and glucose utilization (R(f), R(g); mumol g wet weight(-1) min(-1)) were obtained using 2-deoxy[(3)H]glucose and [(125)I]-15-rho-iodophenyl)-3-R,S-methylpentadecanoic acid. FABP3(+/-) had enhanced cardiac R(g) compared with WT during saline infusion in both LF and HF. FABP3(+/-) abrogated the HF-induced decrement in insulin-stimulated cardiac R(g). On a HF diet, FABP(+/-) but not WT had an increased reliance on fatty acids (R(f)) during insulin stimulation. In conclusion, cardiac insulin resistance and glucose uptake is largely corrected by a reduction in FABP3 in vivo without contemporaneous deleterious effects on cardiac function.

The aim of this study was to determine the contribution of heart-type fatty acid-binding protein (H-FABP) to glucose and long-chain fatty acid (LCFA) utilization in dietary-induced insulin resistance. We tested the hypothesis that H-FABP facilitates increases in LCFA flux present in glucose-intolerant states and that a partial reduction in the amount of this protein would compensate for all or part of the impairment. Transgenic H-FABP heterozygotes (HET) and wild-type (WT) littermates were studied following chow diet (CHD) or high-fat diet (HFD) for 12 weeks. Catheters were surgically implanted in the carotid artery and jugular vein for sampling and infusions, respectively. Following 5 days of recovery, mice received either a saline infusion or underwent a euglycemic insulin clamp (4 mU x kg(-1) x min(-1)) for 120 min. At 90 min, a bolus of 2-deoxyglucose and [125I]-15-(rho-iodophenyl)-3-R,S-methylpentadecanoic acid were administered to obtain indexes of glucose and LCFA utilization. At 120 min, skeletal muscles were excised for tracer determination. All HFD mice were obese and hyperinsulinemic; however, only HFD-WT mice were hyperglycemic. Glucose infusion rates during insulin clamps were 49 +/- 4, 59 +/- 4, 16 +/- 4, and 33 +/- 4 mg x kg(-1) x min(-1) for CHD-WT, CHD-HET, HFD-WT, and HFD-HET mice, respectively, showing that HET limited the severity of whole-body insulin resistance with HFD. Insulin-stimulated muscle glucose utilization was attenuated in HFD-WT but unaffected in HFD-HET mice. Conversely, rates of LCFA clearance were increased with HFD feeding in HFD-WT but not in HFD-HET mice. In conclusion, a partial reduction in H-FABP protein normalizes fasting glucose levels and improves whole-body insulin sensitivity in HFD-fed mice despite obesity.

The infusion of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) causes a rise in tissue concentrations of the AMP analog 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranotide (ZMP), which mimics an elevation of cellular AMP levels. The purpose of this work was to determine the effect of raising hepatic ZMP levels on hepatic insulin action in vivo. Dogs had sampling and infusion catheters as well as flow probes implanted 16 days before an experiment. After an 18-h fast, blood glucose was 82 +/- 1 mg/dl and basal net hepatic glucose output 1.5 +/- 0.2 mg . kg(-1) . min(-1). Dogs received portal venous glucose (3.2 mg . kg(-1) . min(-1)), peripheral venous somatostatin, and basal portal venous glucagon infusions from -90 to 60 min. Physiological hyperinsulinemia was established with a portal insulin infusion (1.2 mU . kg(-1) . min(-1)). Peripheral venous glucose infusion was used to clamp arterial blood glucose at 150 mg/dl. Starting at t = 0 min, dogs received portal venous AICAR infusions of 0, 1, or 2 mg . kg(-1) . min(-1). Net hepatic glucose uptake was 2.4 +/- 0.5 mg . kg(-1) . min(-1) (mean of all groups) before t = 0 min. In the absence of AICAR, net hepatic glucose uptake was 1.9 +/- 0.4 mg . kg(-1) . min(-1) at t = 60 min. The lower-dose AICAR infusion caused a complete suppression of net hepatic glucose uptake (-1.0 +/- 1.7 mg . kg(-1) . min(-1) at t = 60 min). The higher AICAR dose resulted in a profound shift in hepatic glucose balance from net uptake to a marked net output (-6.1 +/- 1.9 mg . kg(-1) . min(-1) at t = 60 min), even in the face of hyperglycemia and hyperinsulinemia. These data show that elevations in hepatic ZMP concentrations, induced by portal venous AICAR infusion, cause acute hepatic insulin resistance. These findings have important implications for the targeting of AMP kinase for the treatment of insulin resistance, using AMP analogs.

Increased plasminogen activator inhibitor 1 (PAI-1) has been linked to not only thrombosis and fibrosis but also to obesity and insulin resistance. Increased PAI-1 levels have been presumed to be consequent to obesity. We investigated the interrelationships of PAI-1, obesity, and insulin resistance in a high-fat/high-carbohydrate (HF) diet-induced obesity model in wild-type (WT) and PAI-1-deficient mice (PAI-1(-/-)). Obesity and insulin resistance developing in WT mice on an HF diet were completely prevented in mice lacking PAI-1. PAI-1(-/-) mice on an HF diet had increased resting metabolic rates and total energy expenditure compared with WT mice, along with a marked increase in uncoupling protein 3 mRNA expression in skeletal muscle, likely mechanisms contributing to the prevention of obesity. In addition, insulin sensitivity was enhanced significantly in PAI-1(-/-) mice on an HF diet, as shown by euglycemic-hyperinsulinemic clamp studies. Peroxisome proliferator-activated receptor (PPAR)-gamma and adiponectin mRNA, key control molecules in lipid metabolism and insulin sensitivity, were maintained in response to an HF diet in white adipose tissue in PAI-1(-/-) mice, contrasting with downregulation in WT mice. This maintenance of PPAR-gamma and adiponectin may also contribute to the observed maintenance of body weight and insulin sensitivity in PAI-1(-/-) mice. Treatment in WT mice on an HF diet with the angiotensin type 1 receptor antagonist to downregulate PAI-1 indeed inhibited PAI-1 increases and ameliorated diet-induced obesity, hyperglycemia, and hyperinsulinemia. PAI-1 deficiency also enhanced basal and insulin-stimulated glucose uptake in adipose cells in vitro. Our data suggest that PAI-1 may not merely increase in response to obesity and insulin resistance, but may have a direct causal role in obesity and insulin resistance. Inhibition of PAI-1 might provide a novel anti-obesity and anti-insulin resistance treatment.

To determine if prior exercise enhances insulin-stimulated extraction of glucose by the liver, chronically catheterized dogs were submitted to 150 min of treadmill exercise or rest. After exercise or rest, dogs received portal glucose (18 micro mol x kg(-1) x min(-1)), peripheral somatostatin, and basal portal glucagon infusions from t = 0 to 150 min. A peripheral glucose infusion was used to clamp arterial blood glucose at 8.3 mmol/l. Insulin was infused into the portal vein to create either basal levels or mild hyperinsulinemia. Prior exercise did not increase whole-body glucose disposal in the presence of basal insulin (25.5 +/- 1.5 vs. 20.3 +/- 1.7 micro mol x kg(-1) x min(-1)), but resulted in a marked enhancement in the presence of elevated insulin (97.2 +/- 15.1 vs. 64.4 +/- 7.4 micro mol x kg(-1) x min(-1)). Prior exercise also increased net hepatic glucose uptake in the presence of both basal insulin (7.5 +/- 1.2 vs. 2.9 +/- 2.4 micro mol x kg(-1) x min(-1)) and elevated insulin (22.0 +/- 3.5 vs. 11.5 +/- 1.8 micro mol x kg(-1) x min(-1)). Likewise, net hepatic glucose fractional extraction was increased by prior exercise with both basal insulin (0.04 +/- 0.01 vs. 0.01 +/- 0.01 micro mol x kg(-1) x min(-1)) and elevated insulin (0.10 +/- 0.01 vs. 0.05 +/- 0.01). Hepatic glycogen synthesis was increased by elevated insulin, but was not enhanced by prior exercise. Although the increase in glucose extraction after exercise could be ascribed to increased insulin action, the increase in hepatic glycogen synthesis was independent of it.

This article addresses two topics. We provide an overview of the National Institutes of Health Mouse Metabolic Phenotyping Center (MMPC) Program. We then discuss some observations we have made during the first eight years of the Vanderbilt MMPC regarding common phenotyping practices. We include specific recommendations to improve phenotyping practices for tests of glucose tolerance and insulin action. We recommend that methods for experiments in vivo be described in manuscripts. We make specific recommendations for data presentation, interpretation, and experimental design for each test. To facilitate and maximize the exchange of scientific information, we suggest that guidelines be developed for methods used to assess glucose tolerance and insulin action in vivo. insulin resistance; insulin clamp; diabetes doi: 10.1152/ajpendo.90996.2008.

We previously showed that global deletion of the cytochrome P450 epoxygenase Cyp2c44, a major epoxyeicosatrienoic acid (EET) producing enzyme in mice, leads to impaired hepatic insulin signaling resulting in insulin resistance. This finding led us to investigate whether administration of a water soluble EET analog restores insulin signaling in vivo in Cyp2c44(-/-) mice and investigated the underlying mechanisms by which this effect is exerted. Cyp2c44(-/-) mice treated with the analog EET-A for 4 weeks improved fasting glucose and glucose tolerance compared to Cyp2c44(-/-) mice treated with vehicle alone. This beneficial effect was accompanied by enhanced hepatic insulin signaling, decreased expression of gluconeogenic genes and increased expression of glycogenic genes. Mechanistically, we show that insulin-stimulated phosphorylation of insulin receptor β (IRβ) is impaired in primary Cyp2c44(-/-) hepatocytes and this can be restored by cotreatment with EET-A and insulin. Plasma membrane fractionations of livers indicated that EET-A enhances the retention of IRβ in membrane rich fractions, thus potentiating its activation. Altogether, EET analogs ameliorate insulin signaling in a genetic model of hepatic insulin resistance by stabilizing membrane-associated IRβ and potentiating insulin signaling. [ABSTRACT FROM AUTHOR]

These studies test, using intravital microscopy (IVM), the hypotheses that perfusion effects on insulin-stimulated muscle glucose uptake (MGU) are 1) capillary recruitment independent and 2) mediated through the dispersion of glucose rather than insulin. For experiment 1, capillary perfusion was visualized before and after intravenous insulin. No capillary recruitment was observed. For experiment 2, mice were treated with vasoactive compounds (sodium nitroprusside, hyaluronidase, and lipopolysaccharide), and dispersion of fluorophores approximating insulin size (10- kDa dextran) and glucose (2-NBDG) was measured using IVM. Subsequently, insulin and 2[14C]deoxyglucose were injected and muscle phospho-2[14C]deoxyglucose (2[C14]DG) accumulation was used as an index of MGU. Flow velocity and 2-NBDG dispersion, but not perfused surface area or 10-kDa dextran dispersion, predicted phospho-2[14C]DG accumulation. For experiment 3, microspheres of the same size and number as are used for contrast-enhanced ultrasound (CEU) studies of capillary recruitment were visualized using IVM. Due to their low concentration, microspheres were present in only a small fraction of blood-perfused capillaries. Microsphereperfused blood volume correlated to flow velocity. These findings suggest that 1) flow velocity rather than capillary recruitment controls microvascular contributions to MGU, 2) glucose dispersion is more predictive of MGU than dispersion of insulin-sized molecules, and 3) CEU measures regional flow velocity rather than capillary recruitment. [ABSTRACT FROM AUTHOR]

In clinical trials, inhibition of cholesteryl ester transfer protein (CETP) raises HDL cholesterol levels but does not robustly improve cardiovascular outcomes. Approximately two-thirds of trial participants are obese. Lower plasma CETP activity is associated with increased cardiovascular risk in human studies, and protective aspects of CETP have been observed in mice fed a high-fat diet (HFD) with regard to metabolic outcomes. To define whether CETP inhibition has different effects depending on the presence of obesity, we performed short-term anacetrapib treatment in chow- and HFD-fed CETP transgenic mice. Anacetrapib raised HDL cholesterol and improved aspects of HDL functionality, including reverse cholesterol transport, and HDL's antioxidative capacity in HFD-fed mice was better than in chow-fed mice. Anacetrapib worsened the anti-inflammatory capacity of HDL in HFD-fed mice. The HDL proteome was markedly different with anacetrapib treatment in HFD- versus chow-fed mice. Despite benefits on HDL, anacetrapib led to liver triglyceride accumulation and insulin resistance in HFD-fed mice. Overall, our results support a physiologic importance of CETP in protecting from fatty liver and demonstrate context selectivity of CETP inhibition that might be important in obese subjects. [ABSTRACT FROM AUTHOR]

Objective Hepatocyte deletion of estrogen receptor alpha (LKO-ERα) worsens fatty liver, dyslipidemia, and insulin resistance in high-fat diet fed female mice. However, whether or not hepatocyte ERα regulates reverse cholesterol transport (RCT) in mice has not yet been reported. Methods and results Using LKO-ERα mice and wild-type (WT) littermates fed a Western-type diet, we found that deletion of hepatocyte ERα impaired in vivo RCT measured by the removal of 3 H-cholesterol from macrophages to the liver, and subsequently to feces, in female mice but not in male mice. Deletion of hepatocyte ERα decreased the capacity of isolated HDL to efflux cholesterol from macrophages and reduced the ability of isolated hepatocytes to accept cholesterol from HDL ex vivo in both sexes. However, only in female mice, LKO-ERα increased serum cholesterol levels and increased HDL particle sizes. Deletion of hepatocyte ERα increased adiposity and worsened insulin resistance to a greater degree in female than male mice. All of the changes lead to a 5.6-fold increase in the size of early atherosclerotic lesions in female LKO-ERα mice compared to WT controls. Conclusions Estrogen signaling through hepatocyte ERα plays an important role in RCT and is protective against lipid retention in the artery wall during early stages of atherosclerosis in female mice fed a Western-type diet. [ABSTRACT FROM AUTHOR]

Diet-induced muscle insulin resistance is associated with expansion of extracellular matrix (ECM) components, such as collagens, and the expression of collagen-binding integrin, α2β1. Integrins transduce signals from ECM via their cytoplasmic domains, which bind to intracellular integrin-binding proteins. The integrin-linked kinase (ILK)-PINCH-parvin (IPP) complex interacts with the cytoplasmic domain of β-integrin subunits and is critical for integrin signaling. In this study we defined the role of ILK, a key component of the IPP complex, in diet-induced muscle insulin resistance. Wild-type (ILK(lox/lox)) and muscle-specific ILK-deficient (ILK(lox/lox)HSAcre) mice were fed chow or a high-fat (HF) diet for 16 weeks. Body weight was not different between ILK(lox/lox) and ILK(lox/lox)HSAcre mice. However, HF-fed ILK(lox/lox)HSAcre mice had improved muscle insulin sensitivity relative to HF-fed ILK(lox/lox) mice, as shown by increased rates of glucose infusion, glucose disappearance, and muscle glucose uptake during a hyperinsulinemic-euglycemic clamp. Improved muscle insulin action in the HF-fed ILK(lox/lox)HSAcre mice was associated with increased insulin-stimulated phosphorylation of Akt and increased muscle capillarization. These results suggest that ILK expression in muscle is a critical component of diet-induced insulin resistance, which possibly acts by impairing insulin signaling and insulin perfusion through capillaries. [ABSTRACT FROM AUTHOR]

Improving mitochondrial oxidant scavenging may be a viable strategy for the treatment of insulin resistance and diabetes. Mice overexpressing the mitochondrial matrix isoform of superoxide dismutase (sod2tg mice) and/or transgenically expressing catalase within the mitochondrial matrix (mcattg mice) have increased scavenging of O2˙ˉ and H2O2, respectively. Furthermore, muscle insulin action is partially preserved in high fat (HF)-fed mcattg mice. The goal of the current study was to test the hypothesis that increased O2˙ˉ scavenging alone or in combination with increased H2O2 scavenging (mtAO mice) enhances in vivo muscle insulin action in the HF-fed mouse. Insulin action was examined in conscious, unrestrained and unstressed wild type (WT), sod2tg, mcattg and mtAO mice using hyperinsulinemic-euglycemic clamps (insulin clamps) combined with radioactive glucose tracers following sixteen weeks of normal chow or HF (60% calories from fat) feeding. Glucose infusion rates, whole body glucose disappearance, and muscle glucose uptake during the insulin clamp were similar in chow- and HF-fed WT and sod2tg mice. Consistent with our previous work, HF-fed mcattg mice had improved muscle insulin action, however, an additive effect was not seen in mtAO mice. Insulin-stimulated Akt phosphorylation in muscle from clamped mice was consistent with glucose flux measurements. These results demonstrate that increased O2˙ˉ scavenging does not improve muscle insulin action in the HF-fed mouse alone or when coupled to increased H2O2 scavenging. [ABSTRACT FROM AUTHOR]

Increased deposition of specific extracellular matrix (ECM) components is a characteristic of insulin-resistant skeletal muscle. Hyaluronan (HA) is a major constituent of the ECM. The hypotheses that 1) HA content is increased in the ECM of insulin-resistant skeletal muscle and 2) reduction of HA in the muscle ECM by long-acting pegylated human recombinant PH20 hyaluronidase (PEGPH20) reverses high-fat (HF) diet--induced muscle insulin resistance were tested. We show that muscle HA was increased in HF diet--induced obese (DIO) mice and that treatment of PEGPH20, which dose-dependently reduced HA in muscle ECM, decreased fat mass, adipocyte size, and hepatic and muscle insulin resistance in DIO mice at 10 mg/kg. Reduced muscle insulin resistance was associated with increased insulin signaling, muscle vascularization, and percent cardiac output to muscle rather than insulin sensitization of muscle per se. Dose-response studies revealed that PEGPH20 dose-dependently increased insulin sensitivity in DIO mice with a minimally effective dose of 0.01 mg/kg. PEGPH20 at doses of 0.1 and 1 mg/kg reduced muscle HA to levels seen in chow-fed mice, decreased fat mass, and increased muscle glucose uptake. These findings suggest that ECM HA is a target for treatment of insulin resistance. [ABSTRACT FROM AUTHOR]

The article presents a study which examined effect of renin overexpression and high-fat feeding on glucose intolerance and angiotensin II (Ang II). The researchers used RenTgMK transgenic mice which are subjected to genotyping, glucose tolerance test, plasma measurement, pancreas histology and immunostaining, stastical analysis and metabolic evaluation. The body weight, fat weight, metabolic levels, insulin resistance and renin-angiotensin system (RAS) reaction are also assessed.

OBJECTIVE--The hypothesis that high-fat (HF) feeding causes skeletal muscle extracellular matrix (ECM) remodeling in C57BL/6J mice and that this remodeling contributes to diet-induced muscle insulin resistance (IE) through the collagen receptor integrin α2β1 was tested. RESEARCH DESIGN AND METHODS--The association between IR and ECM remodeling was studied in mice fed chow or HF diet. Specific genetic and pharmacological murine models were used to study effects of HF feeding on ECM in the absence of IR. The role of ECM-integrin interaction in IR was studied using hyperinsulinemic-euglycemic clamps on integrin α2β1-null (itga2-/-), integrin α2β1-null (itga1-/-), and wild-type littermate mice fed chow or HF. Integrin α2β1 and integrin α2β1 signaling pathways have opposing actions. RESULTS--HF-fed mice had IR and increased muscle collagen (Col) m and ColTV protein; the former was associated with increased transcript, whereas the latter was associated with reduced matrix metalloproteinase 9 activity. Rescue of muscle IR by genetic muscle-specific mitochondria-targeted catalase over-expression or by the phosphodiesterase 5a inhibitor, sildenafil, reversed HF feeding effects on ECM remodeling and increased muscle vascularity. Collagen remained elevated in HF-fed itga2-/- mice. Nevertheless, muscle insulin action and vascularity were increased. Muscle IR in HF-fed itga1-/- mice was unchanged. Insulin sensitivity in chow-fed itga1-/- and itga2-/- mice was not different from wild-type littermates. CONCLUSIONS--ECM collagen expansion is tightly associated with muscle IR. Studies with itga2-/- mice provide mechanistic insight for this association by showing that the link between muscle IR and increased collagen can be uncoupled by the absence of collagen-integrin α2β1 interaction. [ABSTRACT FROM AUTHOR]

Skeletal muscle glucose uptake increases dramatically in response to physical exercise. Moreover, skeletal muscle comprises the vast majority of insulin-sensitive tissue and is a site of dysregulation in the insulin-resistant state. The biochemical and histological composition of the muscle is well defined in a variety of species. However, the functional consequences of muscle biochemical and histological adaptations to physiological and pathophysiological conditions are not well understood. The physiological regulation of muscle glucose uptake is complex. Sites involved in the regulation of muscle glucose uptake are defined by a three-step process consisting of: (1) delivery of glucose to muscle, (2) transport of glucose into the muscle by GLUT4 and (3) phosphorylation of glucose within the muscle by a hexokinase (HK). Muscle blood flow, capillary recruitment and extracellular matrix characteristics determine glucose movement from the blood to the interstitium. Plasma membrane GLUT4 content determines glucose transport into the cell. Muscle HK activity, cellular HK compartmentalization and the concentration of the HK inhibitor glucose 6-phosphate determine the capacity to phosphorylate glucose. Phosphorylation of glucose is irreversible in muscle; therefore, with this reaction, glucose is trapped and the uptake process is complete. Emphasis has been placed on the role of the glucose transport step for glucose influx into muscle with the past assertion that membrane transport is rate limiting. More recent research definitively shows that the distributed control paradigm more accurately defines the regulation of muscle glucose uptake as each of the three steps that define this process are important sites of flux control. [ABSTRACT FROM AUTHOR]

Summary: AS160 has emerged as a key player in insulin-mediated glucose transport through controlling GLUT4 trafficking, which is thought to be regulated by insulin-stimulated phosphorylation of sites including the 14-3-3 binding phospho-Thr649 (equivalent to Thr642 in human AS160). To define physiological roles of AS160-Thr649 phosphorylation and 14-3-3 binding in glucose homeostasis, we substituted this residue by a nonphosphorylatable alanine by knockin mutation in mice. The mutant protein was expressed at normal levels, while insulin-stimulated AS160 binding to 14-3-3s was abolished in homozygous knockin mice. These animals displayed impaired glucose disposal and insulin sensitivity, which were associated with decreased glucose uptake in vivo. Insulin-stimulated glucose transport and cell surface GLUT4 content were reduced in isolated muscles, but not in adipocytes. These results provide genetic evidence that insulin-induced AS160-Thr649 phosphorylation and/or its binding to 14-3-3 play an important role in regulating whole-body glucose homeostasis, at least in part through regulating GLUT4 trafficking in muscle. [ABSTRACT FROM AUTHOR]