AL Rats Research Articles

ANTIOXIDANT AND HISTOLOGIC RESPONSES OF ARTEMETHER-LUMEFANTRINE IN EXPERIMENTALLY COMPROMISED RATS’ TESTES

Aim: The current study assessed the response of MSG and ALon rats' testes histomorphology and homogenate antioxidant markers. Methodology: Thirty (30) male Wistar rats were allotted to six groups(sample size, n=5) by random sampling and exposed to MSG and AL daily and per oral for 7 days thus: Groups A, distilled water (1 mL Kg−1), B, therapeutic concentration of Artemether-lumefantrine, TAL, (1.14 mg Kg−1AL), C, high dose Artemether- lumefantrine, HAL (5.7 mg Kg−1AL), D, MSG (8000 mg Kg−1), E, TAL (1.14 mg Kg−1AL)+ MSG (8000 mg Kg−1) and F, HAL (5.7 mg Kg−1AL)+ MSG (8000 mg Kg−1). Alterations in the testes histology and antioxidant markers were assessed while data were tested for statistical significance by acceptable protocols. Results: MSG mono-therapy altered the rats’ testes anti-oxidation mechanism and histology by increasing (p<0.05) glutathione peroxidase (GPX), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), albumin (ALB), total protein (TP), and magnesium (Mg),but decreasing (p<0.05) zinc (Zn) levels and degenerating spermatids within the seminiferous tubules compared to control, and other treatments (except CAT activity in MSG + HAL-fed group that increased (p<0.05) above that of MSG–fed rats). These responses following TAL and HAL mono-treatments were inconsistent compared to control and MSG mono-therapies. These underscore the proclivity of MSG + HAL co-therapy to up-regulate the apparent dysfunction in CAT metabolism, and the inconsistency in the apparent modulatory responses by AL against effects by MSG mono-therapy in the rats’ testes. Conclusion: AL caused inconsistent modulation of alterations in rats’ testes histology and antioxidant function markers due to MSG assault. The modulation may not be sustainable and the alterations may be instead being spiked warranting caution in co-feeding AL and MSGto rats. Peer Review History: Received 5 December 2023; Revised 28 January 2024; Accepted 29 February; Available online 15 March 2024 Academic Editor: Dr. Amany Mohamed Alboghdadly, Ibn Sina National College for Medical Studies in Jeddah, Saudi Arabia, amanyalboghdadly@gmail.com Average Peer review marks at initial stage: 6.5/10 Average Peer review marks at publication stage: 8.0/10 Reviewers: Dr. Rima Benatoui, Applied Neuroendocrinology Laboratory, Faculty of Sciences, University of Annaba, Algeria. benattouiryma@gmail.com Dr. Tamer Elhabibi, Suez Canal University, Egypt, tamer_hassan@pharm.suez.edu.eg

Independent and combined effects of calorie restriction and AICAR on glucose uptake and insulin signaling in skeletal muscles from 24-month-old female and male rats.

We assessed the effects of two levels of calorie restriction (CR; eating either 15% or 35% less than ad libitum, AL, food intake for 8 weeks) by 24-month-old female and male rats on glucose uptake (GU) and phosphorylation of key signaling proteins (Akt; AMP-activated protein kinase, AMPK; Akt substrate of 160kDa, AS160) measured in isolated skeletal muscles that underwent four incubation conditions (without either insulin or AICAR, an AMPK activator; with AICAR alone; with insulin alone; or with insulin and AICAR). Regardless of sex: (1) neither CR group versus the AL group had greater GU by insulin-stimulated muscles; (2) phosphorylation of Akt in insulin-stimulated muscles was increased in 35% CR versus AL rats; (3) prior AICAR treatment of muscle resulted in greater GU by insulin-stimulated muscles, regardless of diet; and (4) AICAR caused elevated phosphorylation of acetyl CoA carboxylase, an indicator of AMPK activation, in all diet groups. There was a sexually dimorphic diet effect on AS160 phosphorylation, with 35% CR exceeding AL for insulin-stimulated muscles in male rats, but not in female rats. Our working hypothesis is that the lack of a CR-effect on GU by insulin-stimulated muscles was related to the extended duration of the ex vivo incubation period (290 min compared to 40-50 min that was previously reported to be effective). The observed efficacy of prior treatment of muscles with AICAR to improve glucose uptake in insulin-stimulated muscles supports the strategy of targeting AMPK with the goal of improving insulin sensitivity in older females and males.

Astragalus membranaceus Injection Protects Retinal Ganglion Cells by Regulating the Nerve Growth Factor Signaling Pathway in Experimental Rat Traumatic Optic Neuropathy.

Activation of the nerve growth factor (NGF) signaling pathway is a potential method of treatment for retinal ganglion cell (RGC) loss due to traumatic optic neuropathy (TON). The present study aimed to explore the biological effects of injecting Astragalus membranaceus (A. mem) on RGCs in an experimental TON model. Adult male Wistar rats were randomly divided into three groups: sham-operated (SL), model (ML), and A. mem injection (AL). The left eyes of the rats were considered the experimental eyes, and the right eyes served as the controls. AL rats received daily intraperitoneal injections of A. mem (3 mL/kg), whereas ML and SL rats were administered the same volume of normal saline. The TON rat model was induced by optic nerve (ON) transverse quantitative traction. After two-week administration, the number of RGCs was determined using retrograde labeling with Fluoro-Gold. The protein levels of NGF, tyrosine kinase receptor A (TrkA), c-Jun N-terminal protein kinase (JNK), JNK phosphorylation (p-JNK), and nuclear factor kappa-B (NF-κB) were assessed using western blotting. The levels of p75 neurotrophin receptor (p75NTR) and NF-κB DNA binding were examined using real-time PCR and an electrophoretic mobility shift assay. In addition, the concentrations of JNK and p-JNK were assessed using an enzyme-linked immunosorbent assay. Results. The number of RGCs in ML was found to be significantly decreased (P < 0.01) relative to both AL and SL, together with the downregulation of NGF (P < 0.01), TrkA (P < 0.05), and NF-κB (P < 0.01); upregulation of p75NTR mRNA (P < 0.01); and increased protein levels of JNK (P < 0.05) and p-JNK (P < 0.05). Treatment using A. mem injection significantly preserved the density of RGCs in rats with experimental TON and markedly upregulated the proteins of NGF (P < 0.01), TrkA (P < 0.05), and NF-κB (P < 0.01) and downregulated the mRNA level of p75NTR(P < 0.01), as well as the proteins of JNK (P < 0.05) and p-JNK (P < 0.01). Thus, A. mem injection could reduce RGC death in TON induced by ON transverse quantitative traction by stimulating the NGF signaling pathway.

Changes in behavioral and neuronal parameters by alcohol, cigarette, or their combined use in rats.

Few studies have explored the effects of the combined use of alcohol and cigarette in humans, despite its prevalence. Here we evaluated the effect of isolated and combined use on behaviors and neuronal parameters in rats. Male adult rats were divided into alcohol (AL, 2 g/kg, by oral gavage), cigarette smoke (TB, six cigarettes, by inhalation), combined use (ALTB), or control (CT, water by oral gavage and environmental air) groups, treated twice a day (09.00 and 14.00 h). After 4 weeks, the rats were tested in the open field for behavioral analysis and euthanized for brain volume estimation and counting of neurons in the hippocampus. All treatments increased locomotion, and this behavior was higher in the ALTB than TB group. Latency to exit from the central area was lower in the ALTB than in the AL or CT groups. Rearing behavior increased in TB and decreased in AL and ALTB rats. Combined ALTB rats significantly increased their grooming behavior. Only the AL group showed decreased neuron counts and increased brain volume. Our results show that the isolated and combined uses of alcohol and cigarette smoke have diverse effects on behavioral and neuronal parameters in rats after long-term treatment.

Interactions between insulin and diet on striatal dopamine uptake kinetics in rodent brain slices.

Diet influences dopamine transmission in motor- and reward-related basal ganglia circuitry. In part, this reflects diet-dependent regulation of circulating and brain insulin levels. Activation of striatal insulin receptors amplifies axonal dopamine release in brain slices, and regulates food preference invivo. The effect of insulin on dopamine release is indirect, and requires striatal cholinergic interneurons that express insulin receptors. However, insulin also acts directly on dopamine axons to increase dopamine uptake by promoting dopamine transporter (DAT) surface expression, counteracting enhanced dopamine release. Here, we determined the functional consequences of acute insulin exposure and chronic diet-induced changes in insulin on DAT activity after evoked dopamine release in striatal slices from adult ad-libitum fed (AL) rats and mice, and food-restricted (FR) or high-fat/high-sugar obesogenic (OB) diet rats. Uptake kinetics were assessed by fitting evoked dopamine transients to the Michaelis-Menten equationand extracting Cpeak and Vmax . Insulin (30nm) increased both parameters in the caudate putamen and nucleus accumbens core of AL rats in an insulin receptor- and PI3-kinase-dependent manner. A pure effect of insulin on uptake was unmasked using mice lacking striatal acetylcholine, in which increased Vmax caused a decrease in Cpeak . Dietalso influenced Vmax , which was lower in FR vs. AL. The effects of insulin on Cpeak and Vmax were amplified by FR but blunted by OB, consistent with opposite consequences of these diets on insulin levels and insulin receptor sensitivity. Overall, these data reveal acute and chronic effects of insulin and diet on dopamine release and uptake that will influence brain reward pathways.

Effects of Calorie Restriction and Fiber Type on Glucose Uptake and Abundance of Electron Transport Chain and Oxidative Phosphorylation Proteins in Single Fibers from Old Rats.

Calorie restriction (CR; reducing calorie intake by ~40% below ad libitum) can increase glucose uptake by insulin-stimulated muscle. Because skeletal muscle is comprised of multiple, heterogeneous fiber types, our primary aim was to determine the effects of CR (initiated at 14 weeks old) and fiber type on insulin-stimulated glucose uptake by single fibers of diverse fiber types in 23-26-month-old rats. Isolated epitrochlearis muscles from AL and CR rats were incubated with [3H]-2-deoxyglucose ± insulin. Glucose uptake and fiber type were determined for single fibers dissected from the muscles. We also determined CR-effects on abundance of several key metabolic proteins in single fibers. CR resulted in: (a) significantly (p < .05 to .001) greater glucose uptake by insulin-stimulated type I, IIA, IIB, IIBX, and IIX fibers; (b) significantly (p < .05 to .001) reduced abundance of several mitochondrial electron transport chain (ETC) and oxidative phosphorylation (OxPhos) proteins in type I, IIA, and IIBX but not IIB and IIX fibers; and (c) unaltered hexokinase II abundance in each fiber type. These results demonstrate that CR can enhance glucose uptake in each fiber type of rat skeletal muscle in the absence of upregulation of the abundance of hexokinase II or key mitochondrial ETC and OxPhos proteins.

Antinociceptive effects of caloric restriction on post-incisional pain in nonobese rats

Caloric restriction (CR) increases lifespan, retards physiological signs of aging, and delays a variety of diseases. Reduction of inflammatory response was proposed as one of the molecular mechanisms for how CR exerts beneficial effects. The present study investigated the effects of CR on postoperative pain in rats. Adult nonobese rats were divided into two dietary groups, an ad libitum fed group (AL) and a caloric restriction group (CR) that was provided with 60% of the food intake of AL rats. After 6 weeks, the effects of CR on pain behaviors and inflammation induced by plantar incision were examined. CR rats displayed significantly reduced nonevoked pain, mechanical allodynia and thermal hyperalgesia induced by incision, and showed decreased levels of pro-inflammatory cytokines in serum, peri-incisional skin tissue and ipsilateral spinal cord dorsal horn at 6 h and 24 h after incision. The analgesic efficiency of parecoxib and morphine, two agents widely used for the management of postoperative pain clinically, was reinforced by CR. Together, CR generates antinociceptive effects on postoperative incisional pain in rats, perhaps providing some improvement of QOL in patients with postoperative pain, and the beneficial effects may be attributable to the inhibition of excessive inflammation induced by surgical injury.

Activity-Based Anorexia Reduces Body Weight without Inducing a Separate Food Intake Microstructure or Activity Phenotype in Female Rats-Mediation via an Activation of Distinct Brain Nuclei.

Anorexia nervosa (AN) is accompanied by severe somatic and psychosocial complications. However, the underlying pathogenesis is poorly understood, treatment is challenging and often hampered by high relapse. Therefore, more basic research is needed to better understand the disease. Since hyperactivity often plays a role in AN, we characterized an animal model to mimic AN using restricted feeding and hyperactivity. Female Sprague-Dawley rats were divided into four groups: no activity/ad libitum feeding (ad libitum, AL, n = 9), activity/ad libitum feeding (activity, AC, n = 9), no activity/restricted feeding (RF, n = 12) and activity/restricted feeding (activity-based anorexia, ABA, n = 11). During the first week all rats were fed ad libitum, ABA and AC had access to a running wheel for 24 h/day. From week two ABA and RF only had access to food from 9:00 to 10:30 a.m. Body weight was assessed daily, activity and food intake monitored electronically, brain activation assessed using Fos immunohistochemistry at the end of the experiment. While during the first week no body weight differences were observed (p > 0.05), after food restriction RF rats showed a body weight decrease: −13% vs. day eight (p < 0.001) and vs. AC (−22%, p < 0.001) and AL (−26%, p < 0.001) that gained body weight (+10% and +13%, respectively; p < 0.001). ABA showed an additional body weight loss (−9%) compared to RF (p < 0.001) reaching a body weight loss of −22% during the 2-week restricted feeding period (p < 0.001). Food intake was greatly reduced in RF (−38%) and ABA (−41%) compared to AL (p < 0.001). Interestingly, no difference in 1.5-h food intake microstructure was observed between RF and ABA (p > 0.05). Similarly, the daily physical activity was not different between AC and ABA (p > 0.05). The investigation of Fos expression in the brain showed neuronal activation in several brain nuclei such as the supraoptic nucleus, arcuate nucleus, locus coeruleus and nucleus of the solitary tract of ABA compared to AL rats. In conclusion, ABA combining physical activity and restricted feeding likely represents a suited animal model for AN to study pathophysiological alterations and pharmacological treatment options. Nonetheless, cautious interpretation of the data is necessary since rats do not voluntarily reduce their body weight as observed in human AN.

Alterations in energy expenditure in Roux-en-Y gastric bypass rats persist at thermoneutrality.

The compensatory decrease in energy expenditure (EE) in response to body weight loss is attenuated by Roux-en-Y gastric bypass (RYGB) surgery in rats. The thermoneutral zone (TNZ) is at higher temperatures in rodents than in humans. Consequently, rodents may be under moderate cold stress if EE is measured at room temperature, leading to increased EE due to adaptive thermogenesis. We speculated that the reported alterations in EE of RYGB rats at room temperature are caused by higher adaptive thermogenesis and are therefore not present at thermoneutrality. Male Wistar rats were randomized for RYGB or sham surgery. Some sham rats were body weight matched (BWM) to the RYGB rats by food restriction, the others received ad libitum access to food (AL). EE, body temperature, physical activity and food intake were measured at ambient temperatures between 22 and 32 °C to determine the TNZ. Adaptive thermogenesis requires β3-adrenergic receptor-mediated uncoupling protein-1 (UCP-1) expression in brown adipose tissue (BAT). The in vivo thermogenic capacity of BAT was determined by administering the β3-adrenergic agonist CL316,243, and UCP-1 protein expression was measured at room temperature. The TNZ was between 28 and 30 °C for AL and RYGB and between 30 and 32 °C for BWM rats, respectively. In contrast to AL and BWM rats, EE was not significantly higher at room temperature than at thermoneutrality in RYGB rats, reflecting a lack of adaptive thermogenesis. Consistently, both the thermogenic capacity of BAT and UCP-1 expression were decreased in RYGB compared with AL rats at room temperature. Our data confirm that the decrease in EE after body weight loss is attenuated by RYGB surgery and show that this effect persists at thermoneutrality. Contrary to our hypothesis, we found that adaptive thermogenesis at room temperature is reduced in RYGB rats.

Severe Calorie Restriction Reduces Cardiometabolic Risk Factors and Protects Rat Hearts from Ischemia/Reperfusion Injury.

Background and Aims: Recent studies have proposed that if a severe caloric restriction (SCR) is initiated at the earliest period of postnatal life, it can lead to beneficial cardiac adaptations later on. We investigated the effects of SCR in Wistar rats from birth to adult age on risk factors for cardiac diseases (CD), as well as cardiac function, redox status, and HSP72 content in response to ischemia/reperfusion (I/R) injury.Methods and Results: From birth to the age of 3 months, CR50 rats were fed 50% of the food that the ad libitum group (AL) was fed. Food intake was assessed daily and body weight were assessed weekly. In the last week of the SCR protocol, systolic blood pressure and heart rate were measured and the double product index was calculated. Also, oral glucose and intraperitoneal insulin tolerance tests were performed. Thereafter, rats were decapitated, visceral fat was weighed, and blood and hearts were harvested for biochemical, functional, tissue redox status, and western blot analyzes. Compared to AL, CR50 rats had reduced the main risk factors for CD. Moreover, the FR50 rats showed increased cardiac function both at baseline conditions (45% > AL rats) and during the post-ischemic period (60% > AL rats) which may be explained by a decreased cardiac oxidative stress and increased HSP72 content.Conclusion: SCR from birth to adult age reduced risk factors for CD, increased basal cardiac function and protected hearts from the I/R, possibly by a mechanism involving ROS.

Exercise Counters the Changes in Nutrient Sensing Gene Expression after Caloric Restriction and the Loss of Ovarian Function in Obesity‐prone Rats

BackgroundExercise counters the biological drive to regain weight after weight loss, both by reducing the drive to overfeed and by increasing expended energy beyond the cost of the exercise bout. We have hypothesized that exercise alters the nutrient sensing systems in the brain after weight reduction in a manner that would attenuate the drive to overfeed. In the present study, we examined the effect of exercise in female rats after the loss of ovarian function, when the drive to gain weight is known to be exacerbated. We specifically evaluated the gene expression of neural factors involved in glucose sensing and in the regulation of energy balance in the arcuate nucleus (ARC), ventral medial nucleus (VMN), supraoptic nucleus (SON) and amygdala (AMG).MethodDiet induced obese female rats were weight reduced (15% body weight) by restricting caloric intake, with (EX) or without (SED) a daily bout of treadmill exercise (60min/day, 6days/week, 15m/min). The rats were then surgically ovariectomized (OVX), and then allowed ad libitum access for 6 weeks. Food intake, weight gain, and body composition were monitored during this weight gain period. Ad libitum ovariectomized (AL) and sham‐operated (SHAM) rats were included in the design as controls. At the time of sacrifice, punch biopsies of ARC, VMN, SON and AMG were obtained and assayed for gene expression (mRNA) by qPCR. Outcomes included mRNA levels of glucokinase (GK), a hallmark of glucose sensing neurons, glucose transporter 2 and 3 (GLUT2, GLUT3), insulin receptor (InsR), oxytocin (OT), OT receptor (OTR), leptin receptor (LepR), and pro‐opiomelanocortin (POMC), a precursor polypeptide involved in appetite and metabolic regulation.ResultsIn the SON, both GLUT3 and InsR mRNAs were significantly lower in AL and SED rats compared to that in SHAM rats (approximately 40% and 60% lower, respectively), but these levels were restored in EX rats. In the AMG, exercise significantly increased OTR gene expression (about two fold). In the VMN, GK and GLUT3 gene expression decreased by approximately 25% and 30%, respectively in AL rats, and these further decreased in SED rats (~ 50% and 45% reduction, respectively), compared to that in SHAM rats. In both cases, regular exercise tended to restore these levels to that found in the SHAM rats. There was no significant change of GK, GLUT3, Glut2 or POMC mRNA in ARC.ConclusionThe loss of ovarian function altered the expression of critical factors involved in glucose sensing in the SON and VMN in a manner consistent with OVX‐induced overfeeding. Weight reduction through calorie restriction exacerbated some of these effects. Regular exercise, however, countered these combined effects on nutrient sensing gene expression in the SON, VMN, and AMG, and may elicit appetite suppressing effects by enhancing the capacity for and response to nutrients in circulation.Support or Funding InformationNIH P50 HD073063, NIH R01 CA164166, NIH P30 DK48520.

Mild caloric restriction reduces blood pressure and activates endothelial AMPK-PI3K-Akt-eNOS pathway in obese Zucker rats

Genetic obesity models exhibit endothelial dysfunction associated to adenosine monophosphate-activated protein kinase (AMPK) dysregulation. This study aims to assess if mild short-term caloric restriction (CR) restores endothelial AMPK activity leading to an improvement in endothelial function. Twelve-week old Zucker lean and obese (fa/fa) male rats had access to standard chow either ad libitum (AL, n=8) or 80% of AL (CR, n=8) for two weeks. Systolic blood pressure was significantly higher in fa/fa AL rats versus lean AL animals, but was normalized by CR. Endothelium-dependent relaxation to acetylcholine (ACh, 10−9 to 10−4 M) was reduced in fa/fa AL compared to control lean AL rats (p<0.001), and restored by CR. The AMPK activator AICAR (10−5 to 8·10−3 M) elicited a lower relaxation in fa/fa AL rings that was normalized by CR (p<0.001). Inhibition of PI3K (wortmannin, 10−7 M), Akt (triciribine, 10−5 M), or eNOS (L-NAME, 10−4 M) markedly reduced AICAR-induced relaxation in lean AL, but not in fa/fa AL rats. These inhibitions were restored by CR in Zucker fa/fa rings. These data show that mild short-term CR improves endothelial function and lowers blood pressure in obesity due to the activation of the AMPK–PI3K–Akt–eNOS pathway.

Abstract 13780: JNK Inhibition Mimics the Beneficial Effect of Roux-en-Y Gastric Bypass Surgery on Obesity-Induced Endothelial Dysfunction

Introduction: Roux-en-Y gastric bypass surgery (RYGB) reduces weight and long-term cardiovascular risk in obese patients. We previously demonstrated that in diet-induced obese rats RYGB improves aortic endothelial vasorelaxation 8 days after surgery in a weight loss-independent manner. Improved endothelial function was associated with decreased JNK phosphorylation and increased Akt and eNOS phosphorylation in the aorta. Hypothesis: We investigated whether in vivo inhibition of JNK activity in sham-operated ad libitum-fed rats mimics these endothelial effects of RYGB. Methods: Male Wistar rats were fed a high fat (60%) high cholesterol (1.25%) diet for 7 weeks before undergoing either RYGB or sham surgery; sham-operated rats received vehicle (sham_AL) or the JNK-specific inhibitor SP600125 40mg/kg/day s.c. for 8 days post-surgery (sham_SP). Then, thoracic aortic rings were isolated and subjected to ex vivo isometric tension recordings. After submaximal contraction with norepinephrine (10-6mol/L), cumulative relaxation responses were performed to GLP-1 (7-36) amide (10-12 to 10-6mol/L) or insulin (10-12 to 10-5mol/L); tests were repeated after preincubation with the eNOS inhibitor L-NAME (10-4mol/L). Western blot analysis of JNK, Akt and eNOS was also performed on aortic tissue lysates. Results: Body weight did not differ between sham_SP and sham_AL rats, while the weight loss of RYGB rats was significant 8 days after surgery. GLP-1- and insulin-induced vasorelaxation responses improved in RYGB compared to sham_AL rats; interestingly, sham_SP rats had endothelial relaxation similar to RYGB rats. All responses were blocked by preincubation with L-NAME, suggesting an eNOS-dependent effect. JNK protein phosphorylation in aortic lysates was decreased in RYGB and sham_SP rats compared to sham_AL, while Akt phosphorylation was increased. eNOS phosphorylation and dimerization were also increased in RYGB and sham_SP compared to sham_AL rats. Conclusion: JNK inhibition seems to mimic the immediate eNOS-mediated endothelial protective effects of RYGB. Further, our study underlines a crucial role of JNK in obesity-associated endothelial dysfunction, suggesting a novel mechanism for the cardiovascular effects of RYGB.

Diet control to achieve euglycemia induces significant loss of heart and liver weight via increased autophagy compared with ad libitum diet in diabetic rats.

Intensive glucose control increases the all-cause mortality in type 2 diabetes mellitus (T2DM); however, the underlying mechanisms remain unclear. We hypothesized that strict diet control to achieve euglycemia in diabetes damages major organs, increasing the mortality risk. To evaluate effects on major organs when euglycemia is obtained by diet control, we generated a model of end-stage T2DM in 13-week-old Sprague-Dawley rats by subtotal pancreatectomy, followed by ad libitum feeding for 5 weeks. We divided these rats into two groups and for the subsequent 6 weeks provided ad libitum feeding to half (AL, n=12) and a calorie-controlled diet to the other half (R, n=12). To avoid hypoglycemia, the degree of calorie restriction in the R group was isocaloric (g per kg body weight per day) compared with a sham-operated control group (C, n=12). During the 6-week diet control period, AL rats ate three times more than rats in the C or R groups, developing hyperglycemia with renal hyperplasia. R group achieved euglycemia but lost overall body weight significantly compared with the C or AL group (49 or 22%, respectively), heart weight (39 or 23%, respectively) and liver weight (50 or 46%, respectively). Autophagy levels in the heart and liver were the highest in the R group (P<0.01), which also had the lowest pAkt/Akt levels among the groups (P<0.05 in the heart; P<0.01 in the liver). In conclusion, glycemic control achieved by diet control can prevent hyperglycemia-induced renal hyperplasia in diabetes but may be deleterious even at isocaloric rate when insulin is deficient because of significant loss of heart and liver mass via increased autophagy.

Involvement of nucleus accumbens AMPA receptor trafficking in augmentation of D- amphetamine reward in food-restricted rats.

Chronic food restriction (FR) increases behavioral responsiveness to drugs of abuse and associated environments. Pre- and postsynaptic neuroadaptations have been identified in the mesoaccumbens dopamine pathway of FR subjects but the mechanistic basis of increased drug reward magnitude remains unclear. Effects of FR on basal and D-amphetamine-induced trafficking of AMPA receptor subunits to the nucleus accumbens (NAc) postsynaptic density (PSD) were examined, and AMPA receptor involvement in augmentation of D-amphetamine reward was tested. FR and ad libitum fed (AL) rats were injected with D-amphetamine (2.5mg/kg, i.p.) or vehicle. Brains were harvested and subcellular fractionation and Western analyses were used to assess AMPA receptor abundance in NAc homogenate and PSD fractions. A follow-up experiment used a curve-shift protocol of intracranial self-stimulation to assess the effect of 1-naphthylacetyl spermine (1-NASPM), a blocker of Ca(2+)-permeable AMPA receptors, on rewarding effects of D-amphetamine microinjected in NAc shell. FR increased GluA1 in the PSD, and D-amphetamine increased p-Ser845-GluA1, GluA1, GluA2, but not GluA3, with a greater effect in FR than AL rats. D-amphetamine lowered reward thresholds, with greater effects in FR than AL rats, and 1-NASPM selectively reversed the enhancing effect of FR. Results suggest that FR leads to increased synaptic incorporation of GluA1 homomers to potentiate rewarding effects of appetitive stimuli and, as a maladaptive byproduct, D-amphetamine. The D-amphetamine-induced increase in synaptic p-Ser845-GluA1, GluA1, and GluA2 may contribute to the rewarding effect of D-amphetamine, but may also be a mechanism of synaptic strengthening and behavior modification.

Long-term calorie restriction has minimal impact on brain metabolite and fatty acid profiles in aged rats on a Western-style diet

The effect of long-term calorie restriction (CR) on metabolites, fatty acid profiles and energy substrate transporter expression in the brain was assessed in aged rats. Three groups of male Sprague–Dawley rats were studied: (i) a 2month old ad libitum-fed (2AL group), (ii) a 19month old ad libitum-fed (19AL group), and (iii) a 19month old group subjected to 40% CR from the age of 7.5 to 19months (19CR group). The diet contained high sucrose and low n-3 polyunsaturated fatty acids (PUFA) so as to imitate a Western-style diet. High resolution magic angle spinning-1H NMR showed an effect of aging on brain cortex metabolites compared to 2AL rats, the largest differences being for myo-inositol (+251% and +181%), lactate (+203% and +188%), β-hydroxybutyrate (+176% and +618%) and choline (+148% and +120%), in 19AL and 19 CR rats, respectively. However, brain metabolites did not differ between the 19AL and 19CR groups. Cortex fatty acid profiles showed that n-3 PUFA were 35–47% lower but monounsaturated fatty acids were 40–52% higher in 19AL and 19CR rats compared to 2AL rats. Brain microvessel glucose transporter (GLUT1) was 68% higher in 19AL rats than in 2AL rats, while the monocarboxylate transporter, MCT1, was 61% lower in 19CR rats compared to 19AL rats. We conclude that on a high-sucrose, low n-3 PUFA diet, the brain of aged AL rats had higher metabolites and microvessel GLUT1 expression compared to 2AL rats. However, long-term CR in aged rats did not markedly change brain metabolite or fatty acid profile, but did reduce brain microvessel MCT1 expression.

Heterogeneous Effects of Calorie Restriction on In Vivo Glucose Uptake and Insulin Signaling of Individual Rat Skeletal Muscles

Calorie restriction (CR) (consuming ∼60% of ad libitum, AL, intake) improves whole body insulin sensitivity and enhances insulin-stimulated glucose uptake by isolated skeletal muscles. However, little is known about CR-effects on in vivo glucose uptake and insulin signaling in muscle. Accordingly, 9-month-old male AL and CR (initiated when 3-months-old) Fischer 344xBrown Norway rats were studied using a euglycemic-hyperinsulinemic clamp with plasma insulin elevated to a similar level (∼140 µU/ml) in each diet group. Glucose uptake (assessed by infusion of [14C]-2-deoxyglucose, 2-DG), phosphorylation of key insulin signaling proteins (insulin receptor, Akt and Akt substrate of 160kDa, AS160), abundance of GLUT4 and hexokinase proteins, and muscle fiber type composition (myosin heavy chain, MHC, isoform percentages) were determined in four predominantly fast-twitch (epitrochlearis, gastrocnemius, tibialis anterior, plantaris) and two predominantly slow-twitch (soleus, adductor longus) muscles. CR did not result in greater GLUT4 or hexokinase abundance in any of the muscles, and there were no significant diet-related effects on percentages of MHC isoforms. Glucose infusion was greater for CR versus AL rats (P<0.05) concomitant with significantly (P<0.05) elevated 2-DG uptake in 3 of the 4 fast-twitch muscles (epitrochlearis, gastrocnemius, tibialis anterior), without a significant diet-effect on 2-DG uptake by the plantaris or either slow-twitch muscle. Each of the muscles with a CR-related increase in 2-DG uptake was also characterized by significant (P<0.05) increases in phosphorylation of both Akt and AS160. Among the 3 muscles without a CR-related increase in glucose uptake, only the soleus had significant (P<0.05) CR-related increases in Akt and AS160 phosphorylation. The current data revealed that CR leads to greater whole body glucose disposal in part attributable to elevated in vivo insulin-stimulated glucose uptake by fast-twitch muscles. The results also demonstrated that CR does not uniformly enhance either insulin signaling or insulin-stimulated glucose uptake in all muscles in vivo.

Calorie restriction effects on insulin signaling and glucose uptake in multiple insulin‐stimulated rat skeletal muscles in vivo

Calorie restriction (CR; ~60% of ad libitum, AL, food intake) has been shown to enhance whole‐body insulin sensitivity. Skeletal muscle accounts for the majority of insulin‐stimulated glucose disposal, however no previous studies have reported CR effects on both in vivo glucose uptake and insulin signaling in the same skeletal muscles. Accordingly we evaluated the effect of CR (initiated at 14‐wks of age) on the tibialis anterior (TA) and gastrocnemius (GN) muscles from 9‐mo old rats under euglycemic‐hyperinsulinemic clamp conditions. Glucose levels during the clamp were not significantly different for AL (120.7±2.7 mg/dl) versus CR (126.1±2.4 mg/dl) groups. Insulin levels during the clamp were also elevated to similar levels in AL (141.3±9.1 μU/ml) and CR (140.3±6.7 μU/ml) groups. Glucose infusion rates were 78% higher (P&lt;0.05) in CR (31.7±1.6 mg/kg−1 × min−1) versus AL (17.8±0.8 mg/kg−1 × min−1) rats. Glucose uptake (radiolabelled 2‐deoxyglucose) was increased (P&lt;0.05) for CR vs. AL rats in both the TA and GN. Insulin receptorTyr1162/1163 phosphorylation was unaffected by CR in either muscle. AktSer473 phosphorylation was increased (P&lt;0.05) in both muscles of CR vs. AL rats, and there was a strong trend for increased AktThr308 phosphorylation in CR vs. AL rats for both the TA (P=0.052) and GN (P=0.052). These results suggest that CR leads to greater whole‐body insulin sensitivity attributable at least in part to CR effects on in vivo insulin‐stimulated glucose uptake secondary to greater Akt phosphorylation in multiple muscles, including the TA and GN.

Effects of severe caloric restriction from birth on the hearts of adult rats

There has been increasing evidence suggesting that a severe caloric restriction (SCR) (above 40%) has beneficial effects on the hearts of rats. However, most of the reports have focused on the effects of SCR that started in adulthood. We investigated the consequences of SCR on the hearts of rats subjected to SCR since birth (CR50). From birth to the age of 3 months, CR50 rats were fed 50% of the food that the ad libitum group (AL) was fed. Thereafter, a maximal aerobic test was performed to indirectly evaluate global cardiovascular function. Indices of contractility (+dT/dt) and relaxation (-dT/dt) were analyzed in isolated heart preparation, and cardiomyocyte diameter, number, density, and myocardium collagen content were obtained through histologic analysis. Ventricular myocytes were isolated, using standard methods to evaluate phosphorylated AKT levels, and Ca(2+) handling was evaluated with a combination of Western blot analysis, intracellular Ca(2+) imaging, and confocal microscopy. CR50 rats exhibited increased aerobic performance and cardiac function, as shown by the increase in ±dT/dt. Despite the smaller cardiomyocyte diameter, CR50 rats had an increased heart-body weight ratio, increased cardiomyocyte density and number, and similar levels of myocardium collagen content, compared with AL rats. AKT was hyperphosphorylated in cardiomyocytes from CR50 rats, and there were no significant differences in Ca(2+) transient and SERCA2 levels in cardiomyocytes between CR50 and AL rats. Collectively, these observations reveal the beneficial effects of a 50% caloric restriction on the hearts of adult rats restricted since birth, which might involve cardiomyocyte AKT signaling.

Evidence for a metabolic mechanism in the improvement of type 2 diabetes after sleeve gastrectomy in a rodent model

BackgroundSleeve gastrectomy (SG) is at least as effective at producing weight loss as gastric banding but may be superior in producing remission of type 2 diabetes mellitus (T2DM). The objective of this study was to elucidate mechanisms of diabetes improvement in SG beyond caloric restriction. We studied SG in Zucker Diabetic Fatty (ZDF) rats. MethodsTwenty-eight ZDF rats were randomly assigned to 1 of 3 groups: SG, sham-operated ad lib fed (AL), or sham-operated pair fed (PF). SG and AL rats had free access to food. PF rats were fed the average daily intake of the SG group. Comparisons of caloric intake, weight loss, intraperitoneal glucose tolerance testing (IPGTT), insulin, and total ghrelin were performed preoperatively and at postoperative days 10, 20, and 30. Differences between means were evaluated using one-way ANOVA and the paired t test as appropriate. ResultsPostoperatively, SG rats had lower daily caloric intake than the AL controls (78.3±10.5 kcal versus 104.7±4.6 kcal). Both SG and PF groups had sustained weight loss (–5.3±3.8 g and –27.5±2.6 g, respectively); however, SG rats had significantly lower AUC for glucose after IPGTT than both controls. This is in contrast to AL controls that experienced weight gain (34.1±4.7 g) and increases in AUC for glucose after IPGTT. ConclusionAlthough SG is considered a restrictive procedure, there is evidence for a metabolic effect by virtue of decreased insulin resistance, which may not be reproduced by PF controls.