Optimized wound closure using a biomechanical abdominal model.

BACKGROUND: Suturing techniques for midline abdominal wall incisions vary between surgeons. This study uses a biomechanical abdominal model to assess tissue stretch using different suturing techniques for midline laparotomy closure. METHODS: Deformation tests were performed on the linea alba of 48 porcine abdominal walls. Each pattern was tested three times at pressures ranging from 0 to 20 kPa using different continuous suturing techniques and a control. RESULTS: There was a sevenfold improvement when the best performing bite separation and bite width ([5, 16] mm) was compared with the most poorly performing combination ([15, 4] mm). The traditional bite and width separation ([10, 10] mm) and the recently proposed combination ([5, 5] mm) may not be optimal, and substantial improvements in surgical outcome may be achieved by changing to a [5,16]-mm combination. CONCLUSION: These findings suggest using a small bite separation (5 mm) and large bite width (16 mm) during abdominal wound closure may be optimal. Surgical relevance Suturing techniques for midline abdominal wall incisions vary between surgeons. This experimental study suggests substantial potential for improved tissue apposition by changing the suturing approach from the traditional clinical recommendation of 10 mm for both bite separation and bite width to a bite separation of 5 mm and a bite width of 16 mm. These findings support recent European Hernia Society guidelines and the recent randomized STITCH (Suture Techniques to Reduce the Incidence of The inCisional Hernia) trial, which found that small separations are more effective than large separations, but suggest that they should be combined with large bite depths.

Exploring the relationship between α-actinin-3 deficiency and obesity in mice and humans.

Obesity is a worldwide health crisis, and the identification of genetic modifiers of weight gain is crucial in understanding this complex disorder. A common null polymorphism in the fast fiber-specific gene ACTN3 (R577X) is known to influence skeletal muscle function and metabolism. α-Actinin-3 deficiency occurs in an estimated 1.5 billion people worldwide, and results in reduced muscle strength and a shift towards a more efficient oxidative metabolism. The X-allele has undergone strong positive selection during recent human evolution, and in this study, we sought to determine whether ACTN3 genotype influences weight gain and obesity in mice and humans. An Actn3 KO mouse has been generated on two genetic backgrounds (129X1/SvJ and C57BL/6J) and fed a high-fat diet (HFD, 45% calories from fat). Anthropomorphic features (including body weight) were examined and show that Actn3 KO 129X1/SvJ mice gained less weight compared to WT. In addition, six independent human cohorts were genotyped for ACTN3 R577X (Rs1815739) and body mass index (BMI), waist-to-hip ratio-adjusted BMI (WHRadjBMI) and obesity-related traits were assessed. In humans, ACTN3 genotype alone does not contribute to alterations in BMI or obesity.

Mouse strain-dependent variation in obesity and glucose homeostasis in response to high-fat feeding.

AIMS/HYPOTHESIS: Metabolic disorders are commonly investigated using knockout and transgenic mouse models. A variety of mouse strains have been used for this purpose. However, mouse strains can differ in their inherent propensities to develop metabolic disease, which may affect the experimental outcomes of metabolic studies. We have investigated strain-dependent differences in the susceptibility to diet-induced obesity and insulin resistance in five commonly used inbred mouse strains (C57BL/6J, 129X1/SvJ, BALB/c, DBA/2 and FVB/N). METHODS: Mice were fed either a low-fat or a high-fat diet (HFD) for 8 weeks. Whole-body energy expenditure and body composition were then determined. Tissues were used to measure markers of mitochondrial metabolism, inflammation, oxidative stress and lipid accumulation. RESULTS: BL6, 129X1, DBA/2 and FVB/N mice were all susceptible to varying degrees to HFD-induced obesity, glucose intolerance and insulin resistance, but BALB/c mice exhibited some protection from these detrimental effects. This protection could not be explained by differences in mitochondrial metabolism or oxidative stress in liver or muscle, or inflammation in adipose tissue. Interestingly, in contrast with the other strains, BALB/c mice did not accumulate excess lipid (triacylglycerols and diacylglycerols) in the liver; this is potentially related to lower fatty acid uptake rather than differences in lipogenesis or lipid oxidation. CONCLUSIONS/INTERPRETATION: Collectively, our findings indicate that most mouse strains develop metabolic defects on an HFD. However, there are inherent differences between strains, and thus the genetic background needs to be considered carefully in metabolic studies.

Distinct patterns of tissue-specific lipid accumulation during the induction of insulin resistance in mice by high-fat feeding.

AIMS/HYPOTHESIS: While it is well known that diet-induced obesity causes insulin resistance, the precise mechanisms underpinning the initiation of insulin resistance are unclear. To determine factors that may cause insulin resistance, we have performed a detailed time-course study in mice fed a high-fat diet (HFD). METHODS: C57Bl/6 mice were fed chow or an HFD from 3 days to 16 weeks and glucose tolerance and tissue-specific insulin action were determined. Tissue lipid profiles were analysed by mass spectrometry and inflammatory markers were measured in adipose tissue, liver and skeletal muscle. RESULTS: Glucose intolerance developed within 3 days of the HFD and did not deteriorate further in the period to 12 weeks. Whole-body insulin resistance, measured by hyperinsulinaemic-euglycaemic clamp, was detected after 1 week of HFD and was due to hepatic insulin resistance. Adipose tissue was insulin resistant after 1 week, while skeletal muscle displayed insulin resistance at 3 weeks, coinciding with a defect in glucose disposal. Interestingly, no further deterioration in insulin sensitivity was observed in any tissue after this initial defect. Diacylglycerol content was increased in liver and muscle when insulin resistance first developed, while the onset of insulin resistance in adipose tissue was associated with increases in ceramide and sphingomyelin. Adipose tissue inflammation was only detected at 16 weeks of HFD and did not correlate with the induction of insulin resistance. CONCLUSIONS/INTERPRETATION: HFD-induced whole-body insulin resistance is initiated by impaired hepatic insulin action and exacerbated by skeletal muscle insulin resistance and is associated with the accumulation of specific bioactive lipid species.

Differential regulation of adaptive and apoptotic unfolded protein response signalling by cytokine-induced nitric oxide production in mouse pancreatic beta cells.

AIMS/HYPOTHESIS: Pro-inflammatory cytokines such as IL-1ß, IFN-γ and TNF-α may contribute to pancreatic beta cell destruction in type 1 diabetes. A mechanism requiring nitric oxide, which is generated by inducible nitric oxide synthase (iNOS), in cytokine-induced endoplasmic reticulum (ER) stress and apoptosis has been proposed. Here, we tested the role of nitric oxide in cytokine-induced ER stress and the subsequent unfolded protein response (UPR) in beta cells. METHODS: Isolated islets from wild-type and iNos (also known as Nos2) knockout (iNos ( -/- )) mice, and MIN6 beta cells were incubated with IL-1ß, IFN-γ and TNF-α for 24-48 h. N (G)-methyl-L: -arginine was used to inhibit nitric oxide production in MIN6 cells. Protein levels and gene expression were assessed by western blot and real-time RT-PCR. RESULTS: In islets and MIN6 cells, inhibition of nitric oxide production had no effect on the generation of ER stress by cytokines, as evidenced by downregulation of Serca2b (also known as Atp2a2) mRNA and increased phosphorylation of PKR-like ER kinase, Jun N-terminal kinase (JNK) and eukaryotic translation initiation factor 2 α subunit. However, nitric oxide regulated the pattern of UPR signalling, which delineates the cellular decision to adapt to ER stress or to undergo apoptosis. Inhibition of nitric oxide production led to reduced expression of pro-apoptotic UPR markers, Chop (also known as Ddit3), Atf3 and Trib3. In contrast, adaptive UPR markers (chaperones, foldases and degradation enhancers) were increased. Further analysis of mouse islets showed that cytokine-induced Chop and Atf3 expression was also dependent on JNK activity. CONCLUSIONS/INTERPRETATION: The mechanism by which cytokines induce ER stress in mouse beta cells is independent of nitric oxide production. However, nitric oxide may regulate the switch between adaptive and apoptotic UPR signalling.

Amelioration of lipid-induced insulin resistance in rat skeletal muscle by overexpression of Pgc-1ß involves reductions in long-chain acyl-CoA levels and oxidative stress.

AIMS/HYPOTHESIS: To determine if acute overexpression of peroxisome proliferator-activated receptor, gamma, coactivator 1 beta (Pgc-1ß [also known as Ppargc1b]) in skeletal muscle improves insulin action in a rodent model of diet-induced insulin resistance. METHODS: Rats were fed either a low-fat or high-fat diet (HFD) for 4 weeks. In vivo electroporation was used to overexpress Pgc-1ß in the tibialis cranialis (TC) and extensor digitorum longus (EDL) muscles. Downstream effects of Pgc-1ß on markers of mitochondrial oxidative capacity, oxidative stress and muscle lipid levels were characterised. Insulin action was examined ex vivo using intact muscle strips and in vivo via a hyperinsulinaemic-euglycaemic clamp. RESULTS: Pgc-1ß gene expression was increased >100% over basal levels. The levels of proteins involved in mitochondrial function, lipid metabolism and antioxidant defences, the activity of oxidative enzymes, and substrate oxidative capacity were all increased in muscles overexpressing Pgc-1ß. In rats fed a HFD, increasing the levels of Pgc-1ß partially ameliorated muscle insulin resistance, in association with decreased levels of long-chain acyl-CoAs (LCACoAs) and increased antioxidant defences. CONCLUSIONS: Our data show that an increase in Pgc-1ß expression in vivo activates a coordinated subset of genes that increase mitochondrial substrate oxidation, defend against oxidative stress and improve lipid-induced insulin resistance in skeletal muscle.

Overexpression of the orphan receptor Nur77 alters glucose metabolism in rat muscle cells and rat muscle in vivo.

AIMS/HYPOTHESIS: A hallmark feature of the metabolic syndrome is abnormal glucose metabolism which can be improved by exercise. Recently the orphan nuclear receptor subfamily 4, group A, member 1 (NUR77) was found to be induced by exercise in muscle and was linked to transcriptional control of genes involved in lipid and glucose metabolism. Here we investigated if overexpression of Nur77 (also known as Nr4a1) in skeletal muscle has functional consequences for lipid and/or glucose metabolism. METHODS: L6 rat skeletal muscle myotubes were infected with a Nur77-coding adenovirus and lipid and glucose oxidation was measured. Nur77 was also overexpressed in skeletal muscle of chow- and fat-fed rats and the effects on glucose and lipid metabolism evaluated. RESULTS: Nur77 overexpression had no effect on lipid oxidation in L6 cells or rat muscle, but did increase glucose oxidation and glycogen synthesis in L6 cells. In chow- and high-fat-fed rats, Nur77 overexpression by electrotransfer significantly increased basal glucose uptake and glycogen synthesis, but no increase in insulin-stimulated glucose metabolism was observed. Nur77 electrotransfer was associated with increased production of GLUT4 and glycogenin and increased hexokinase and phosphofructokinase activity. Interestingly, Nur77 expression in muscle biopsies from obese men was significantly lower than in those from lean men and was closely correlated with body-fat content and insulin sensitivity. CONCLUSIONS/INTERPRETATION: Our data provide compelling evidence that NUR77 is a functional regulator of glucose metabolism in skeletal muscle in vivo. Importantly, the diminished content in muscle of obese insulin-resistant men suggests that it might be a potential therapeutic target for the treatment of dysregulated glucose metabolism.

The Ski proto-oncogene regulates body composition and suppresses lipogenesis.

OBJECTIVE: The Ski gene regulates skeletal muscle differentiation in vitro and and in vivo. In the c-Ski overexpression mouse model there occurs marked skeletal muscle hypertrophy with decreased adipose tissue mass. In this study, we have investigated the underlying molecular mechanisms responsible for the increased skeletal muscle and decreased adipose tissue mass in the c-Ski mouse. APPROACH: Growth and body composition analysis (tissue weights and dual energy X-ray absorptiometry) coupled with skeletal muscle and white adipose gene expression and metabolic phenotyping in c-Ski mice and wild-type (WT) littermate controls was performed. RESULTS: The growth and body composition studies confirmed the early onset of accelerated body growth, with increased lean mass and decreased fat mass in the c-Ski mice. Gene expression analysis in skeletal muscle from c-Ski mice compared with WT mice showed significant differences in myogenic and lipogenic gene expressions that are consistent with the body composition phenotype. Skeletal muscle of c-Ski mice had significantly repressed Smad1, 4, 7 and myostatin gene expression and elevated myogenin, myocyte enhancer factor 2, insulin-like growth factor-1 receptor and insulin-like growth factor-2 expression. Strikingly, expression of the mRNAs encoding the master lipogenic regulators, sterol-regulatory enhancer binding protein 1c (SREBP1c), and the nuclear receptor liver X-receptor-alpha, and their downstream target genes, SCD-1 and FAS, were suppressed in skeletal muscle of c-Ski mice, as were the expressions of other nuclear receptors involved in adipogenesis and metabolism, such as peroxisome proliferator-activated receptor-gamma, glucocorticoid receptor and retinoic acid receptor-related orphan receptor-alpha. Transfection analysis demonstrated Ski repressed the SREBP1c promoter. Moreover, palmitate oxidation and oxidative enzyme activity was increased in skeletal muscle of c-Ski mice. These results suggest that the Ski phenotype involves attenuated lipogenesis, decreased myostatin signalling, coupled to increased myogenesis and fatty acid oxidation. CONCLUSION: Ski regulates several genetic programs and signalling pathways that regulate skeletal muscle and adipose mass to influence body composition development, suggesting that Ski may have a role in risk for obesity and metabolic disease.

Diverse roles for protein kinase C delta and protein kinase C epsilon in the generation of high-fat-diet-induced glucose intolerance in mice: regulation of lipogenesis by protein kinase C delta.

AIMS/HYPOTHESIS: This study aimed to determine whether protein kinase C (PKC) delta plays a role in the glucose intolerance caused by a high-fat diet, and whether it could compensate for loss of PKCepsilon in the generation of insulin resistance in skeletal muscle. METHODS: Prkcd (-/-), Prkce (-/-) and wild-type mice were fed high-fat diets and subjected to glucose tolerance tests. Blood glucose levels and insulin responses were determined during the tests. Insulin signalling in liver and muscle was assessed after acute in vivo insulin stimulation by immunoblotting with phospho-specific antibodies. Activation of PKC isoforms in muscle from Prkce (-/-) mice was assessed by determining intracellular distribution. Tissues and plasma were assayed for triacylglycerol accumulation, and hepatic production of lipogenic enzymes was determined by immunoblotting. RESULTS: Both Prkcd (-/-) and Prkce (-/-) mice were protected against high-fat-diet-induced glucose intolerance. In Prkce (-/-) mice this was mediated through enhanced insulin availability, while in Prkcd (-/-) mice the reversal occurred in the absence of elevated insulin. Neither the high-fat diet nor Prkcd deletion affected maximal insulin signalling. The activation of PKCdelta in muscle from fat-fed mice was enhanced by Prkce deletion. PKCdelta-deficient mice exhibited reduced liver triacylglycerol accumulation and diminished production of lipogenic enzymes. CONCLUSIONS/INTERPRETATION: Deletion of genes encoding isoforms of PKC can improve glucose intolerance, either by enhancing insulin availability in the case of Prkce, or by reducing lipid accumulation in the case of Prkcd. The absence of PKCepsilon in muscle may be compensated by increased activation of PKCdelta in fat-fed mice, suggesting that an additional role for PKCepsilon in this tissue is masked.

Mentalising skills in generic mental healthcare settings: can we make our day-to-day interactions more therapeutic?

Aims and methodCaring for patients with personality disorder is one of the biggest challenges in psychiatric work. We investigated whether mentalisation-based treatment skills (MBT-S) teaching improves clinicians' understanding of mentalising and attitudes towards personality disorder. Self-report questionnaires (Knowledge and Application of MBT (KAMQ) and Attitudes to Personality Disorder (APDQ)) were completed at baseline and after a 2-day MBT-S workshop. RESULTS: Ninety-two healthcare professionals completed questionnaires before and after training. The mean within-participant increase in scores from baseline to end-of-programme was 11.6 points (95% CI 10.0-13.3) for the KAMQ and 4.0 points (1.8-6.2) for the APDQ.Clinical implicationsMBT-S is a short intervention that is effective in improving clinicians' knowledge of personality disorder and mentalisation. That attitudes to personality disorder improved overall is encouraging in relation to the possibility of deeper learning in staff and, ultimately, improved care for patients with personality disorder.Declaration of interestNone.

Novel outreach settings to enhance sexually transmitted infection/HIV awareness, diagnosis and treatment in hard-to-reach populations.

Despite recent rises in the number of cases of sexually transmitted infections (STIs) such as syphilis and gonorrhoea in England and increasing rates of HIV diagnosis among several men who have sex with men populations, many individuals are still not engaging with sexual health services. The John Hunter Clinic for Sexual Health, Chelsea and Westminster Hospital, London set up outreach clinics at the two world's largest adult lifestyle exhibitions in 2013 and 2015. This was the first time that a sexual health screening and promotion service was available at these large-scale (over 10,000 attendees at each) adult lifestyle events. A total of 381 individuals underwent STI screening across the two events. Nineteen (5.0%) patients were diagnosed with an infection. Twelve (3.1%) patients with Chlamydia trachomatis, three (0.8%) patients with syphilis, one (0.3%) patient with Neisseria gonorrhoeae, one (0.3%) patient with HIV, one (0.3%) patient with hepatitis B and one (0.3%) patient with hepatitis C. All 19 patients were promptly contacted with their results and had arrangements made for treatment or were referred for specialist follow up. Where possible, contact tracing was also performed. Implementing such outreach-based projects is challenged by lack of on-site laboratory support, high staffing demands and potentially high costs. However, we achieved a total HIV screening uptake rate of 94.5% amongst our outreach clinic attendees (versus 67% nationally in conventional sexual health clinic attendees) with an HIV positivity rate of 0.3% (versus 0.2% nationally in high HIV prevalence band populations). Additionally, 30.7% had never been tested for HIV previously (versus 20.7% nationally). Our work demonstrates that these strategies can help to address issues related to lack of STI/HIV screening in hard-to-reach populations and promote risk reduction behaviour.

Adrenalectomy reduces neuropeptide Y-induced insulin release and NPY receptor expression in the rat ventromedial hypothalamus.

Chronic central administration of neuropeptide Y (NPY) causes hyperphagia, hyperinsulinemia, and obesity, a response that is prevented by prior adrenalectomy (ADX) in rats. The basis of NPY's effect and how the acute responses to this peptide are affected by ADX remain unknown. This study investigates the role of glucocorticoids in acute NPY-stimulated food intake, acute NPY-induced insulin release, and hypothalamic NPY-receptor mRNA expression levels. NPY-induced food intake was similar in ADX and control rats after acute intracerebroventricular injection of NPY. Injection of NPY caused a significant increase in plasma insulin in control rats, but this effect was completely absent in ADX rats in which basal plasma insulin levels were also lower than controls. In addition, ADX significantly reduced the number of neurons expressing NPY receptor Y(1) and Y(5) mRNAs in the ventromedial hypothalamus (VMH), without affecting Y(1)- or Y(5)-mRNA expression in the paraventricular hypothalamus or the arcuate nucleus. These data indicate that glucocorticoids are necessary for acute NPY-mediated insulin release and suggest that the mechanisms involve glucocorticoid regulation of Y(1) and Y(5) receptors specifically within the VMH nucleus.

Differences in lipogenesis in tissues of control and gold-thioglucose obese mice after an isocaloric meal.

Lipogenesis was measured in 2 and 5 week gold-thioglucose (GTG) obese mice after a single meal of 0.5 g of standard chow. Compared to control mice the rate of lipogenesis in GTG obese mice, was 4-fold higher in liver and 10-fold higher in white adipose tissue (WAT). In brown adipose tissue (BAT) of GTG-injected mice the lipogenic rate was only 50% of that of controls. These results indicate that the increased lipid synthesis observed in GTG-injected mice is not due solely to hyperphagia and that some other stimuli, such as increased basal insulin levels and/or decreased thermogenesis and insulin resistance in BAT, contribute to the high rates of fat synthesis in this animal model of obesity.

Acyl-CoA inhibition of hexokinase in rat and human skeletal muscle is a potential mechanism of lipid-induced insulin resistance.

There are strong correlations between impaired insulin-stimulated glucose metabolism and increased intramuscular lipid pools; however, the mechanism by which lipids interact with glucose metabolism is not completely understood. Long-chain acyl CoAs have been reported to allosterically inhibit liver glucokinase (hexokinase IV). The aim of the present study was to determine whether long-chain acyl CoAs inhibit hexokinase in rat and human skeletal muscle. At subsaturating glucose concentrations, 10 micromol/l of the three major long-chain acyl-CoA species in skeletal muscle, palmitoyl CoA (16:0), oleoyl CoA (18:1, n = 9), and linoleoyl CoA (18:2, n = 6), reduced hexokinase activity of rat skeletal muscle to 61 +/- 3, 66 +/- 7, and 57 +/- 5% of control activity (P < 0.005), respectively. The inhibition was concentration-dependent (P < 0.005) with 5 pmol/l producing near maximal inhibition. Human skeletal muscle hexokinase was also inhibited by long-chain acyl CoAs (5 pmol/l palmitoyl CoA decreased activity to 75 +/- 6% of control activity, P < 0.005). Inhibition of hexokinase in rat and human muscle by long-chain acyl CoAs was additive to the inhibition of hexokinase by glucose-6-phosphate (an allosteric inhibitor of hexokinase). This inhibition of skeletal muscle hexokinase by long-chain acyl CoA suggests that increases in intramuscular lipid metabolites could interact directly with insulin-mediated glucose metabolism in vivo by decreasing the rate of glucose phosphorylation and decreasing glucose-6-phosphate concentrations.

Insulin response to an intravenous glucose load during development of obesity in gold thioglucose-injected mice.

The insulin secretory response to an intravenous glucose load was examined in chronically catheterized, conscious mice 2, 5, and 10 wk after induction of obesity by a single injection of gold thioglucose. At 2 wk after administration of gold thioglucose, a significant increase in both the insulinemia and incremental area under the curve of insulin release after intravenous glucose were observed (incremental area under the curve for 2-wk control mice, 852 +/- 54 min/pM; incremental area under the curve for 2-wk GTG-injected mice, 1140 +/- 114 min/pM; P < 0.05). At this stage, no significant difference existed in the glucose tolerance or body weight of control and gold thioglucose-injected mice. By 5 wk, the gold thioglucose-injected mice were approximately 33% heavier than their lean controls and showed a marked glucose intolerance. This was accompanied by overt hyperinsulinemia in both the basal state and also in response to an intravenous glucose bolus as indicated by the increase in the incremental area under the curve of insulin (5-wk control mice, 816 +/- 114 min/pM; 5-wk gold thioglucose-injected mice, 1374 +/- 156 min/pM; P < 0.05). At 10 wk after gold thioglucose administration, body weight and the degree of glucose intolerance were increased. Although 10-wk gold thioglucose-injected mice showed basal hyperinsulinemia, an intravenous glucose bolus elicited a smaller insulin secretory response than that observed in the age-matched lean control animals (10-wk control mice, 672 +/- 54 min/pM; 10-wk gold-thioglucose-injected mice 186 +/- 42 min/pM; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Amelioration of high-fat feeding-induced insulin resistance in skeletal muscle with the antiglucocorticoid RU486.

Fat feeding produces whole-body insulin resistance and decreased glucose uptake in muscle tissue of rats. To examine the effect of glucocorticoid blockade on the insulin resistance caused by high-fat feeding, four groups of rats were fed diets high in starch (70% of calories) or fat (59% of calories) for 4 weeks with or without the antiglucocorticoid RU486 (69.8 mumol.kg-1.day-1) in the food. Whole-body insulin action was assessed by the euglycemic clamp technique at an upper physiological insulin level with bolus 2-[3H]deoxyglucose to determine individual tissue insulin-stimulated glucose uptake. Whole-body glucose utilization (clamp glucose infusion rate [GIR]) was decreased by high-fat feeding (GIR 68.3 +/- 12.2 vs. 182.6 +/- 12.8 mumol.kg-1.min-1 for the starch-fed group; P < 0.001). Addition of RU486 to the diet significantly improved (GIR 133.9 +/- 12.8 mumol.kg-1.min-1; P < 0.01), but did not fully reverse, the insulin resistance caused by fat feeding. RU486 was without effect in the starch-fed rats. In skeletal muscles, RU486 ameliorated 62 and 68% of the insulin resistance produced by fat feeding in red quadriceps and extensor digitorum longus hindlimb muscles, respectively, but had no effect in heart or white adipose tissue. These results suggest that glucocorticoids play, in a tissue-specific manner, a role in the maintenance and/or production of insulin resistance produced by high-fat feeding.

Insulin response in individual tissues of control and gold thioglucose-obese mice in vivo with [1-14C]2-deoxyglucose.

The dose-response characteristics of several glucose-utilizing tissues (brain, heart, white adipose tissue, brown adipose tissue, and quadriceps muscle) to a single injection of insulin have been compared in control mice and mice made obese with a single injection of gold thioglucose (GTG). Tissue content of [1-14C]2-deoxyglucose 6-phosphate and blood disappearance rate of [1-14C]2-deoxyglucose (2-DG) were measured at nine different insulin doses and used to calculate rates of 2-DG uptake and phosphorylation in tissues from control and obese mice. The insulin sensitivity of tissues reflected in the ED50 of insulin response varied widely, and brown adipose tissue was the most insulin-sensitive tissue studied. In GTG-obese mice, heart, quadriceps, and brown adipose tissue were insulin resistant (demonstrated by increased ED50), whereas in white adipose tissue, 2-DG phosphorylation was more sensitive to insulin. Brain 2-DG phosphorylation was insulin independent in control and obese animals. The largest decrease in insulin sensitivity in GTG-obese mice was observed in brown adipose tissue. The loss of diet-induced thermogenesis in brown adipose tissue as a result of the hypothalamic lesion in GTG-obese mice could be a major cause of insulin resistance in brown adipose tissue. Because brown adipose tissue can make a major contribution to whole-body glucose utilization, insulin resistance in this tissue may have a significant effect on whole-animal glucose homeostasis in GTG-obese mice.

Mechanisms of liver and muscle insulin resistance induced by chronic high-fat feeding.

To elucidate cellular mechanisms of insulin resistance induced by excess dietary fat, we studied conscious chronically high-fat-fed (HFF) and control chow diet-fed rats during euglycemic-hyperinsulinemic (560 pmol/l plasma insulin) clamps. Compared with chow diet feeding, fat feeding significantly impaired insulin action (reduced whole body glucose disposal rate, reduced skeletal muscle glucose metabolism, and decreased insulin suppressibility of hepatic glucose production [HGP]). In HFF rats, hyperinsulinemia significantly suppressed circulating free fatty acids but not the intracellular availability of fatty acid in skeletal muscle (long chain fatty acyl-CoA esters remained at 230% above control levels). In HFF animals, acute blockade of beta-oxidation using etomoxir increased insulin-stimulated muscle glucose uptake, via a selective increase in the component directed to glycolysis, but did not reverse the defect in net glycogen synthesis or glycogen synthase. In clamp HFF animals, etomoxir did not significantly alter the reduced ability of insulin to suppress HGP, but induced substantial depletion of hepatic glycogen content. This implied that gluconeogenesis was reduced by inhibition of hepatic fatty acid oxidation and that an alternative mechanism was involved in the elevated HGP in HFF rats. Evidence was then obtained suggesting that this involves a reduction in hepatic glucokinase (GK) activity and an inability of insulin to acutely lower glucose-6-phosphatase (G-6-Pase) activity. Overall, a 76% increase in the activity ratio G-6-Pase/GK was observed, which would favor net hepatic glucose release and elevated HGP in HFF rats. Thus in the insulin-resistant HFF rat 1) acute hyperinsulinemia fails to quench elevated muscle and liver lipid availability, 2) elevated lipid oxidation opposes insulin stimulation of muscle glucose oxidation (perhaps via the glucose-fatty acid cycle) and suppression of hepatic gluconeogenesis, and 3) mechanisms of impaired insulin-stimulated glucose storage and HGP suppressibility are not dependent on concomitant lipid oxidation; in the case of HGP we provide evidence for pivotal involvement of G-6-Pase and GK in the regulation of HGP by insulin, independent of the glucose source.

Peroxisome proliferator-activated receptor (PPAR)-alpha activation lowers muscle lipids and improves insulin sensitivity in high fat-fed rats: comparison with PPAR-gamma activation.

Peroxisome proliferator-activated receptor (PPAR)-alpha agonists lower circulating lipids, but the consequences for muscle lipid metabolism and insulin sensitivity are not clear. We investigated whether PPAR-alpha activation improves insulin sensitivity in insulin-resistant rats and compared the effects with PPAR-gamma activation. Three-week high fat-fed male Wistar rats were untreated or treated with the specific PPAR-alpha agonist WY14643 or the PPAR-gamma agonist pioglitazone (both 3 mg x kg(-1) x day(-1)) for the last 2 weeks of high-fat feeding. Like pioglitazone, WY14643 lowered basal plasma levels of glucose, triglycerides (-16% vs. untreated), and leptin (-52%), and also muscle triglyceride (-34%) and total long-chain acyl-CoAs (LCACoAs) (-41%) (P < 0.05). In contrast to pioglitazone, WY14643 substantially reduced visceral fat weight and total liver triglyceride content (P < 0.01) without increasing body weight gain. WY14643 and pioglitazone similarly enhanced whole-body insulin sensitivity (clamp glucose infusion rate increased 35 and 37% and glucose disposal 22 and 15%, respectively, vs. untreated). Both agents enhanced insulin-mediated muscle glucose metabolic index (Rg') and reduced muscle triglyceride and LCACoA accumulation (P < 0.05). Although pioglitazone had more potent effects than WY14643 on muscle insulin sensitization, this was associated with its greater effect to reduce muscle LCACoA accumulation. Overall insulin-mediated muscle Rg' was inversely correlated with the content of LCACoAs (r = -0.74, P = 0.001) and with plasma triglyceride levels (r = -0.77, P < 0.001). We conclude that even though WY14643 and pioglitazone, representing PPAR-alpha and PPAR-gamma activation, respectively, may alter muscle lipid supply by different mechanisms, both significantly improve muscle insulin action in the high fat-fed rat model of insulin resistance, and this effect is proportional to the degree to which they reduce muscle lipid accumulation.

Local factors modulate tissue-specific NEFA utilization: assessment in rats using 3H-(R)-2-bromopalmitate.

Insulin-resistant states are associated with accumulation of muscle lipid, suggesting an imbalance between lipid uptake and oxidation. We have employed a new fatty-acid tracer [9,10-3H]-(R)-2-bromopalmitate (3H-R-BrP) to study individual-tissue nonesterified fatty acid (NEFA) uptake in states with diminished or enhanced lipid oxidation. 3H-R-BrP was administered to conscious male Wistar rats (approximately 300 g) during fasting (5, 18, or 36 h), acute blockade of beta-oxidation (etomoxir, 15 micromol/kg), and insulin infusion (0.25 U x kg(-1) x h(-1)). Estimates of NEFA clearance rates (K(f)*) and absolute rates of uptake (R(f)*) were calculated from tissue accumulation of 3H-R-BrP products. In the basal state, NEFA uptake was dependent on the oxidative capacity of tissues: R(f)* in brown adipose tissue (BAT) > heart (HRT) > diaphragm (DPHM) > red quadriceps (RQ) > white quadriceps (WQ) > white adipose tissue (WAT). Fasting increased (P < 0.001) K(f)* in WAT but did not change NEFA clearance in other tissues. However, plasma NEFA levels were raised (P < 0.01), tending to elevate R(f)* in most tissues (P < 0.05: WAT, BAT, WQ, DPHM). Etomoxir reduced (P < 0.01) K(f)* only in oxidative tissues (BAT, RQ, DPHM, HRT). Insulin lowered plasma NEFA levels (P < 0.001) and significantly decreased R(f)* in most tissues (P < 0.05: WAT, RQ, DPHM, HRT). An increased (P < 0.05) clearance was observed in WAT, BAT, and WQ; a decrease (P < 0.01) in K(f)* was observed in HRT. This study is the first to measure tissue-specific NEFA uptake in conscious rats in the postabsorptive, fasted, and insulin-stimulated states. We have demonstrated that tissue NEFA utilization is not exclusively determined by systemic availability, but that the early steps of NEFA uptake or metabolic sequestration can also be rapidly modulated by local processes such as NEFA oxidation.