Insulin action on protein synthesis and its association with eIF5A expression and hypusination.

The hormone insulin plays a central role in the metabolism of carbohydrates, lipids, and proteins. In relation to protein metabolism, insulin stimulates amino acid uptake and activates protein synthesis in responsive cells by modulation of signal transduction pathways, such as associated to Akt/PkB, mTOR, S6Ks, 4E-BP1, and several translation initiation/elongation factors. In this context, there is no information on direct cellular treatment with insulin and effects on eukaryotic translation initiation factor 5A (eIF5A) regulation. The eIF5A protein contains an exclusive amino acid residue denominated hypusine, which is essential for its activity and synthesized by posttranslational modification of a specific lysine residue using spermidine as substrate. The eIF5A protein is involved in cellular proliferation and differentiation processes, as observed for satellite cells derived from rat muscles, revealing that eIF5A has an important role in muscle regeneration. The aim of this study was to determine whether eIF5A expression and hypusination are influenced by direct treatment of insulin on L6 myoblast cells. We observed that insulin increased the content of eIF5A transcripts. This effect occurred in cells treated or depleted of fetal bovine serum, revealing a positive insulin effect independent of other serum components. In addition, it was observed that hypusination follows the maintenance of eIF5A protein content in the serum depleted cells and treated with insulin. These results demonstrate that eIF5A is modulated by insulin, contributing the protein synthesis machinery control, as observed by puromycin incorporation in nascent proteins.

Combined treatment with melatonin and insulin improves glycemic control, white adipose tissue metabolism and reproductive axis of diabetic male rats.

AIMS: Melatonin treatment has been reported to be capable of ameliorating metabolic diabetes-related abnormalities but also to cause hypogonadism in rats. We investigated whether the combined treatment with melatonin and insulin can improve insulin resistance and other metabolic disorders in rats with streptozotocin-induced diabetes during neonatal period and the repercussion of this treatment on the hypothalamic-pituitary-gonadal axis. MAIN METHODS: At the fourth week of age, diabetic animals started an 8-wk treatment with only melatonin (0.2 mg/kg body weight) added to drinking water at night or associated with insulin (NHP, 1.5 U/100 g/day) or only insulin. Animals were then euthanized, and the subcutaneous (SC), epididymal (EP), and retroperitoneal (RP) fat pads were excised, weighed and processed for adipocyte isolation for morphometric analysis as well as for measuring glucose uptake, oxidation, and incorporation of glucose into lipids. Hypothalamus was collected for gene expression and blood samples were collected for biochemical assays. KEY FINDINGS: The treatment with melatonin plus insulin (MI) was capable of maintaining glycemic control. In epididymal (EP) and subcutaneous (SC) adipocytes, the melatonin plus insulin (MI) treatment group recovered the insulin responsiveness. In the hypothalamus, melatonin treatment alone promoted a significant reduction in kisspeptin-1, neurokinin B and androgen receptor mRNA levels, in relation to control group. SIGNIFICANCE: Combined treatment with melatonin and insulin promoted a better glycemic control, improving insulin sensitivity in white adipose tissue (WAT). Indeed, melatonin treatment reduced hypothalamic genes related to reproductive function.

Alternative Start Codon Connects eIF5A to Mitochondria.

Eukaryotic translation initiation factor 5A (eIF5A), a protein containing the amino acid residue hypusine required for its activity, is involved in a number of physiological and pathological cellular processes. In humans, several EIF5A1 transcript variants encode the canonical eIF5A1 isoform B, whereas the hitherto uncharacterized variant A is expected to code for a hypothetical eIF5A1 isoform, referred to as isoform A, which has an additional N-terminal extension. Herein, we validate the existence of eIF5A1 isoform A and its production from transcript variant A. In fact, variant A was shown to encode both eIF5A1 isoforms A and B. Mutagenic assays revealed different efficiencies in the start codons present in variant A, contributing to the production of isoform B at higher levels than isoform A. Immunoblotting and mass spectrometric analyses showed that isoform A can undergo hypusination and acetylation at specific lysine residues, as observed for isoform B. Examination of the N-terminal extension suggested that it might confer mitochondrial targeting. Correspondingly, we found that isoform A, but not isoform B, co-purified with mitochondria when the proteins were overproduced. These findings suggest that eIF5A1 isoform A has a role in mitochondrial function. J. Cell. Physiol. 231: 2682-2689, 2016. © 2016 Wiley Periodicals, Inc.

Metabolic adaptations in the adipose tissue that underlie the body fat mass gain in middle-aged rats.

Little is known about adipocyte metabolism during aging process and whether this can influence body fat redistribution and systemic metabolism. To better understand this phenomenon, two animal groups were studied: young-14 weeks old-and middle-aged-16 months old. Periepididymal (PE) and subcutaneous (SC) adipocytes were isolated and tested for their capacities to perform lipolysis and to incorporate D-[U-(14)C]-glucose, D-[U-(14)C]-lactate, and [9,10(n)-(3)H]-oleic acid into lipids. Additionally, the morphometric characteristics of the adipose tissues, glucose tolerance tests, and biochemical determinations (fasting glucose, triglycerides, insulin) in blood were performed. The middle-aged rats showed adipocyte (PE and SC) hypertrophy and glucose intolerance, although there were no significant changes in fasting glycemia and insulin. Furthermore, PE tissue revealed elevated rates (+50 %) of lipolysis during beta-adrenergic-stimulation. There was also an increase (+62 %) in the baseline rate of glucose incorporation into lipids in the PE adipocytes, while these PE cells were almost unresponsive to insulin stimulation and less responsive (a 34 % decrease) in the SC tissue. Also, the capacity of oleic acid esterification was elevated in baseline state and with insulin stimulus in the PE tissue (+90 and 82 %, respectively). Likewise, spontaneous incorporation of lactate into lipids in the PE and SC tissues was higher (+100 and 11 %, respectively) in middle-aged rats. We concluded that adipocyte metabolism of middle-aged animals seems to strongly favor cellular hypertrophy and increased adipose mass, particularly the intra-abdominal PE fat pad. In discussion, we have interpreted all these results as a metabolic adaptations to avoid the spreading of fat that can reach tissues beyond adipose protecting them against ectopic fat accumulation. However, these adaptations may have the potential to lead to future metabolic dysfunctions seen in the senescence.

Effects of cafeteria diet on the jejunum in sedentary and physically trained rats.

OBJECTIVE: The effects of a cafeteria diet on the small intestine were investigated in adult Wistar rats under sedentary conditions and after physical training. METHODS: Parameters including morphometry, enzyme activities, and total myenteric populations in the jejunum were evaluated. RESULTS: The cafeteria diet, characterized as hyperlipidic, produced obese rats, corroborated by increases in the Lee index and the weights of the periepididymal and retroperitoneal adipose tissues (P<0.01). Obesity caused increases in the length of the small intestine, villi height, crypt depth, whole-wall thickness (P<0.05), and the enzymatic activities of alkaline phosphatase, lipase, and sucrase (P<0.01), in addition to a reduction in the number of goblet cells (P<0.05). With reference to the jejunal intrinsic innervations, the total number and area of myenteric neurons was unchanged regardless of the group. Physical training promoted 1) a reduction of the weight in the retroperitoneal and periepididymal adipose tissues (P<0.05) and 2) an increase in the thickness of the muscular layer (P<0.05). CONCLUSION: The cafeteria diet promoted obesity in rodents, leading to alterations in morphometry and enzymatic intestinal parameters, which were partily attenuated by physical training.