Constitutively Active CaMKKα Stimulates Skeletal Muscle Glucose Uptake in Insulin-Resistant Mice In Vivo

In insulin-sensitive skeletal muscle, the expression of constitutively active Ca²⁺/calmodulin-dependent protein kinase kinase α (caCaMKKα) stimulates glucose uptake independent of insulin signaling (i.e., Akt and Akt-dependent TBC1D1/TBC1D4 phosphorylation). Our objectives were to determine whether caCaMKKα could stimulate glucose uptake additively with insulin in insulin-sensitive muscle, in the basal state in insulin-resistant muscle, and if so, to determine whether the effects were associated with altered TBC1D1/TBC1D4 phosphorylation. Mice were fed a control or high-fat diet (60% kcal) for 12 weeks to induce insulin resistance. Muscles were transfected with empty vector or caCaMKKα plasmids using in vivo electroporation. After 2 weeks, caCaMKKα protein was robustly expressed. In insulin-sensitive muscle, caCaMKKα increased basal in vivo [³H]-2-deoxyglucose uptake approximately twofold, insulin increased glucose uptake approximately twofold, and caCaMKKα plus insulin increased glucose uptake approximately fourfold. caCaMKKα did not increase basal TBC1D1 (Ser²³⁷, Thr⁵⁹⁰, Ser⁶⁶⁰, pan-Thr/Ser) or TBC1D4 (Ser⁵⁸⁸, Thr⁶⁴², pan-Thr/Ser) phosphorylation. In insulin-resistant muscle, caCaMKKα increased basal glucose uptake approximately twofold, and attenuated high-fat diet–induced basal TBC1D1 (Thr⁵⁹⁰, pan-Thr/Ser) and TBC1D4 (Ser⁵⁸⁸, Thr⁶⁴², pan-Thr/Ser) phosphorylation. In cell-free assays, CaMKKα increased TBC1D1 (Thr⁵⁹⁰, pan-Thr/Ser) and TBC1D4 (Ser⁵⁸⁸, pan-Thr/Ser) phosphorylation. Collectively, these results demonstrate that caCaMKKα stimulates glucose uptake additively with insulin, and in insulin-resistant muscle, and alters the phosphorylation of TBC1D1/TBC1D4.     

Adiponectin Corrects High-Fat Diet–Induced Disturbances in Muscle Metabolomic Profile and Whole-Body Glucose Homeostasis

We provide here a detailed and comprehensive analysis of skeletal muscle metabolomic profiles in response to adiponectin in adiponectin knockout (AdKO) mice after high-fat–diet (HFD) feeding. Hyperinsulinemic-euglycemic clamp studies showed that adiponectin administration corrected HFD-induced defects in post/basal insulin stimulated R_d and insulin signaling in skeletal muscle. Lipidomic profiling of skeletal muscle from HFD-fed mice indicated elevated triacylglycerol and diacylglycerol species (16:0–18:1, 18:1, and 18:0–18:2) as well as acetyl coA, all of which were mitigated by adiponectin. HFD induced elevated levels of various ceramides, but these were not significantly altered by adiponectin. Adiponectin corrected the altered branched-chain amino acid metabolism caused by HFD and corrected increases across a range of glycerolipids, fatty acids, and various lysolipids. Adiponectin also reversed induction of the pentose phosphate pathway by HFD. Analysis of muscle mitochondrial structure indicated that adiponectin treatment corrected HFD-induced pathological changes. In summary, we show an unbiased comprehensive metabolomic profile of skeletal muscle from AdKO mice subjected to HFD with or without adiponectin and relate these to changes in whole-body glucose handling, insulin signaling, and mitochondrial structure and function. Our data revealed a key signature of relatively normalized muscle metabolism across multiple metabolic pathways with adiponectin supplementation under the HFD condition.Adiponectin circulates abundantly in the concentration range of 3–30 µg/mL in healthy subjects (1). Decreased plasma adiponectin, in particular the high–molecular weight (HMW) oligomeric form, has been found in patients with obesity and type 2 diabetes despite increasing adipose tissue mass (2). Numerous studies in humans or animal models have consistently shown that adiponectin can mediate antidiabetes effects via insulin-sensitizing or insulin-mimetic effects in various tissues (3). However, the precise influence of adiponectin on a wide range of metabolic variables has not been fully characterized.Skeletal muscle serves as a major site for glucose and fatty acid metabolism (4). The development of insulin resistance owing to obesity is a multifactorial vicious cycle whereby various mechanisms interact with each other to worsen the condition over time (5). In lean/healthy individuals, skeletal muscle has the capacity to switch between carbohydrate and lipid as the preferred energy substrate, and this phenomenon is called metabolic flexibility (6). This flexibility is lost in individuals with insulin resistance and mitochondrial dysfunction. Mitochondrial dysfunction in skeletal muscle is considered central to the pathogenesis of insulin resistance and metabolic complications in obesity and type 2 diabetes (7,8). In obesity, mitochondrial function must be maximized to efficiently handle the prolonged overload of energy substrates—mainly lipids. However, decreased oxidative capacity or incomplete oxidation (6) in dysfunctional mitochondria contributes to the accumulation of lipid intermediates such as ceramide and diacylglycerol (DAG) inside the myocyte, which leads to insulin resistance (9–11). Evidence points to an important contribution of adiponectin availability or action in such metabolic adaptations. For example, studies using adiponectin knockout (AdKO) mice have been particularly informative, as these mice displayed more severe insulin resistance than their wild-type (wt) counterparts when challenged with a high-fat high-sucrose diet (12). Consistent with these findings, a recent publication from Kadowaki and colleagues (13) showed that adiponectin can increase muscle mitochondrial mass and its oxidative capacity by the activation of AMP kinase (14). Furthermore, transgenic mice overexpressing adiponectin show improved mitochondrial function and insulin sensitivity (15–17).Although it is well established that adiponectin exerts beneficial metabolic effects by enhancing insulin sensitivity, further investigations need to be conducted to better understand the underlying molecular mechanisms. Metabolomics is now emerging as a powerful tool for providing a precise functional profile of cellular biochemistry (18). In this way, novel discoveries connecting metabolic profiles and biological responses have been made and have expedited advances in our understanding of cell biology and physiology in all areas of medicine, including diabetes (19–21).We performed this study using AdKO mice to explore the impacts of dietary high-fat content and adiponectin supplementation on global metabolic profiles in skeletal muscle and correlate these with biochemical and histological data as well as measures of insulin sensitivity and whole-body glucose homeostasis. The study was designed to further test the hypothesis that restoring adiponectin action alleviates the metabolic deterioration in muscle metabolism and whole-body glucose handling elicited by long-term high-fat feeding. To this end, AdKO mice were fed a high-fat diet (HFD) and treated daily with adiponectin to restore levels seen in wt, chow-fed mice. The study focused on identification of novel modulators of muscle metabolism through metabolomic analysis.     

Skeletal muscle as an endocrine organ: PGC-1α, myokines and exercise

An active lifestyle is crucial to maintain health into old age; inversely, sedentariness has been linked to an elevated risk for many chronic diseases. The discovery of myokines, hormones produced by skeletal muscle tissue, suggests the possibility that these might be molecular mediators of the whole body effects of exercise originating from contracting muscle fibers. Even though less is known about the sedentary state, the lack of contraction-induced myokines or the production of a distinct set of hormones in the inactive muscle could likewise contribute to pathological consequences in this context. In this review, we try to summarize the most recent developments in the study of muscle as an endocrine organ and speculate about the potential impact on our understanding of exercise and sedentary physiology, respectively.This article is part of a Special Issue entitled "Muscle Bone Interactions".     

C-Reactive Protein Causes Insulin Resistance in Mice Through Fcγ Receptor IIB–Mediated Inhibition of Skeletal Muscle Glucose Delivery

Elevations in C-reactive protein (CRP) are associated with an increased risk of insulin resistance. Whether CRP plays a causal role is unknown. Here we show that CRP transgenic mice and wild-type mice administered recombinant CRP are insulin resistant. Mice lacking the inhibitory Fcγ receptor IIB (FcγRIIB) are protected from CRP-induced insulin resistance, and immunohistochemistry reveals that FcγRIIB is expressed in skeletal muscle microvascular endothelium and is absent in skeletal muscle myocytes, adipocytes, and hepatocytes. The primary mechanism in glucose homeostasis disrupted by CRP is skeletal muscle glucose delivery, and CRP attenuates insulin-induced skeletal muscle blood flow. CRP does not impair skeletal muscle glucose delivery in FcγRIIB^−/− mice or in endothelial nitric oxide synthase knock-in mice with phosphomimetic modification of Ser1176, which is normally phosphorylated by insulin signaling to stimulate nitric oxide–mediated skeletal muscle blood flow and glucose delivery and is dephosphorylated by CRP/FcγRIIB. Thus, CRP causes insulin resistance in mice through FcγRIIB-mediated inhibition of skeletal muscle glucose delivery.Numerous clinical studies indicate that chronic, mild increases in circulating levels of the acute-phase reactant C-reactive protein (CRP) are associated with insulin resistance (1–5). For example, in middle-aged men, independent of numerous risk factors including baseline BMI, individuals in the top quintile of CRP (>4.18 μg/mL) had a more than threefold greater risk of developing diabetes than those in the lowest quintile (<0.66 μg/mL) (6). However, the relationship between CRP and type 2 diabetes has been greatly debated (7–10), and whether CRP plays a pathogenetic role is unknown.In the current study, we determined how CRP influences glucose homeostasis in vivo, testing the hypothesis that CRP induces insulin resistance in mice. Additional studies were performed to address the following questions:

• Which regulatory processes in glucose homeostasis are altered by CRP?
• Is the effect of CRP on glucose homeostasis mediated by Fcγ receptors (FcγR) for IgG, which bind CRP to invoke its cellular actions in certain paradigms (11,12)?
• How does CRP action via FcγR cause insulin resistance?

     

Role of PKCδ in Insulin Sensitivity and Skeletal Muscle Metabolism

Protein kinase C (PKC)δ has been shown to be increased in liver in obesity and plays an important role in the development of hepatic insulin resistance in both mice and humans. In the current study, we explored the role of PKCδ in skeletal muscle in the control of insulin sensitivity and glucose metabolism by generating mice in which PKCδ was deleted specifically in muscle using Cre-lox recombination. Deletion of PKCδ in muscle improved insulin signaling in young mice, especially at low insulin doses; however, this did not change glucose tolerance or insulin tolerance tests done with pharmacological levels of insulin. Likewise, in young mice, muscle-specific deletion of PKCδ did not rescue high-fat diet–induced insulin resistance or glucose intolerance. However, with an increase in age, PKCδ levels in muscle increased, and by 6 to 7 months of age, muscle-specific deletion of PKCδ improved whole-body insulin sensitivity and muscle insulin resistance and by 15 months of age improved the age-related decline in whole-body glucose tolerance. At 15 months of age, M-PKCδKO mice also exhibited decreased metabolic rate and lower levels of some proteins of the OXPHOS complex suggesting a role for PKCδ in the regulation of mitochondrial mass at older age. These data indicate an important role of PKCδ in the regulation of insulin sensitivity and mitochondrial homeostasis in skeletal muscle with aging.     

Decreased Hypoxia-Inducible Factor-1α in Gastrocnemius Muscle in Rats with Chronic Kidney Disease

Background/Aims: Hypoxia-inducible factor (HIF)-1α is responsible for increased expression of genes engaged in angiogenesis. Our previous study indicated capillary rarefaction and atrophy of glycolytic fibers, mainly in locomotor muscles of uremic animals. Perhaps these changes are secondary to disturbances of HIF-1α in skeletal muscles. Methods: Expression of HIF-1α at mRNA and protein levels, as well as mRNA of vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS), in gastrocnemius muscle (MG) and longissimus thoracic muscle (ML) were measured by RT-PCR and Western blot. Rats were randomized to subtotal nephrectomy (CKD5/6), uninephrectomy (CKD1/2) or sham operation (controls). Results: For CKD5/6 versus controls, mRNA levels for HIF-1α, VEGF-A, VEGFR-1 and VEGFR-2 were significantly reduced only in MG, while eNOS was significantly decreased and iNOS was significantly increased only in ML. Western blot analysis indicated significantly increased HIF-1α protein levels in MG and ML from CKD1/2 animals versus controls, whereas in the CKD5/6 group, the level of HIF-1α protein decreased significantly in MG and increased significantly in ML versus controls and CKD1/2. Conclusion: The reduced expression of HIF-1α mRNA and protein in locomotor muscle from CKD5/6 animals may be involved in the pathogenesis of uremic myopathy. Increased expression of iNOS in the postural muscles may act as a protective factor through HIF-1α stabilization.     

Heat-induced changes in the expression and localisation of PGC-1α in the mice testis

The functions of mammalian testis are temperature-sensitive. There are various testicular factors, which express in response to heat as a mechanism of defence. PGC-1α and HSP70 have poetical role in the protection from oxidative stress in various tissues, including testis. The expression of PGC-1α and HSP70 has been shown in the testis, and it has also been documented that heat modulates the expression of PGC-1α and HSP70. However, heat-dependent changes in the localisation and expression of PGC-1α have not been investigated so far. Thus, we studied the expression and localisation pattern of PGC-1α in the testis of heat-treated mice along with marker of proliferation (PCNA, GCNA), serum testosterone levels, MDA levels and HSP70. The results showed a significant increase in PGC-1α and HSP70 and MDA levels in the testis of heat-treated mice along with a decrease in PCNA, GCNA and serum testosterone levels. The immunolocalisation study showed intense immunostaining of PGC-1α in the Leydig cell and germ cells of the heat-treated testis, with pronounced damaged in the histoarchitecture. The results showed that increase expression of PGC-1α in germ cells and Leydig cells of testis could be a counter mechanism to cope up with oxidative stress in coordination with HSP70.     

Duodenal–Jejunal Bypass Improves Glucose Homeostasis in Association with Decreased Proinflammatory Response and Activation of JNK in the Liver and Adipose Tissue in a T2DM Rat Model

Background

There is accumulating evidence that obesity leads to a proinflammatory state, which plays crucial roles in insulin resistance and development of type 2 diabetes mellitus (T2DM). Previous studies demonstrated that weight loss after bariatric surgery was accompanied by a suppression of the proinflammatory state. However, the effect of bariatric surgery on the proinflammatory state and associated signaling beyond weight loss is still elusive. The objective of this study was to investigate the effect of duodenal–jejunal bypass (DJB) on glucose homeostasis, the proinflammatory state and the involving signaling independently of weight loss.

Methods

A high-fat diet and low-dose streptozotocin administration were used to induce T2DM in male Sprague–Dawley rats. The diabetic rats underwent DJB or sham surgery. The blood glucose, glucose tolerance and insulin resistance were determined to evaluate the glucose homeostasis. Serum insulin, GLP-1 and hsCRP were detected by ELISA. The gene expression of TNF-α, IL-6, IL-1β and MCP-1 in liver and fat was determined by quantitative real-time RT-PCR. The JNK activity and serine phosphorylation of IRS-1 in liver and adipose tissue were determined by Western blotting.

Results

Compared to the S-DJB group, DJB induced significant and sustained glycemic control with improved insulin sensitivity and glucose tolerance independently of weight loss. DJB improved the proinflammatory state indicated by decreased circulating hsCRP and proinflammatory gene expression in the liver and adipose tissue. The JNK activity and serine phosphorylation of IRS-1 in liver and adipose tissue were significantly reduced after DJB.

Conclusions

DJB achieved a rapid and sustainable glycemic control independently of weight loss. The data indicated that the improved proinflammatory state and decreased JNK activity after DJB may contribute to the improved glucose homeostasis.     

Duodenal�CJejunal Bypass Surgery Does Not Increase Skeletal Muscle Insulin Signal Transduction or Glucose Disposal in Goto�CKakizaki Type 2 Diabetic Rats

Background

Duodenal?Cjejunal bypass (DJB) has been shown to reverse type 2 diabetes (T2DM) in Goto?CKakizaki (GK) rats, a rodent model of non-obese T2DM. Skeletal muscle insulin resistance is a hallmark decrement in T2DM. The aim of the current work was to investigate the effects of DJB on skeletal muscle insulin signal transduction and glucose disposal. It was hypothesized that DJB would increase skeletal muscle insulin signal transduction and glucose disposal in GK rats.

Methods

DJB was performed in GK rats. Sham operations were performed in GK and nondiabetic Wistar?CKyoto (WKY) rats. At 2 weeks post-DJB, oral glucose tolerance (OGTT) was measured. At 3 weeks post-DJB, insulin-induced signal transduction and glucose disposal were measured in skeletal muscle.

Results

In GK rats and compared to sham operation, DJB did not (1) improve fasting glucose or insulin, (2) improve OGTT, or (3) increase skeletal muscle insulin signal transduction or glucose disposal. Interestingly, skeletal muscle glucose disposal was similar between WKY-Sham, GK-Sham, and GK-DJB.

Conclusions

Bypassing of the proximal small intestine does not increase skeletal muscle glucose disposal. The lack of skeletal muscle insulin resistance in GK rats questions whether this animal model is adequate to investigate the etiology and treatments for T2DM. Additionally, bypassing of the foregut may lead to different findings in other animal models of T2DM as well as in T2DM patients.     

Is Intestinal Gluconeogenesis a Key Factor in the Early Changes in Glucose Homeostasis Following Gastric Bypass?

Background  

In 2008, Troy et al. hypothesised that under fasting conditions, intestinal gluconeogenesis generates glucose levels in the portal vein which trigger the portal sensor to change insulin resistance and that this mechanism contributes to the effects of Roux-en-Y gastric bypass (RYGB) surgery on type 2 diabetes mellitus (T2DM). In a recent paper, Kashyap et al. (Int J Obes 34(3):426–471, 2010) cited this hypothesis as a potential explanation for the early changes in insulin sensitivity and beta cell function seen after RYGB. We proposed a study to examine this possibility.     

Glucagon-Like Peptide 1 Recruits Muscle Microvasculature and Improves Insulin’s Metabolic Action in the Presence of Insulin Resistance

Glucagon-like peptide 1 (GLP-1) acutely recruits muscle microvasculature, increases muscle delivery of insulin, and enhances muscle use of glucose, independent of its effect on insulin secretion. To examine whether GLP-1 modulates muscle microvascular and metabolic insulin responses in the setting of insulin resistance, we assessed muscle microvascular blood volume (MBV), flow velocity, and blood flow in control insulin-sensitive rats and rats made insulin-resistant acutely (systemic lipid infusion) or chronically (high-fat diet [HFD]) before and after a euglycemic-hyperinsulinemic clamp (3 mU/kg/min) with or without superimposed systemic GLP-1 infusion. Insulin significantly recruited muscle microvasculature and addition of GLP-1 further expanded muscle MBV and increased insulin-mediated glucose disposal. GLP-1 infusion potently recruited muscle microvasculature in the presence of either acute or chronic insulin resistance by increasing muscle MBV. This was associated with an increased muscle delivery of insulin and muscle interstitial oxygen saturation. Muscle insulin sensitivity was completely restored in the presence of systemic lipid infusion and significantly improved in rats fed an HFD. We conclude that GLP-1 infusion potently expands muscle microvascular surface area and improves insulin’s metabolic action in the insulin-resistant states. This may contribute to improved glycemic control seen in diabetic patients receiving incretin-based therapy.