Adaptations of skeletal muscle pyruvate dehydrogenase kinase in response to food-restriction in mitochondrial subpopulations

University, 2006 Dr. Sandra J. Peters Pyruvate dehydrogenase (PDH) catalyses the decarboxylation of pyruvate, to form acetyl-CoA. PDH activity is down-regulated by intrinsic PDH kinases (predominantly PDK2 and PDK4 isoforms), but the understanding of the PDK isoform distribution and adaptation to nutritional stresses has been restricted to mixed mitochondrial populations, and not delineated between subsarcolemmal (SS) and intermyofibrillar (IMF) subpopulations. SS and IMF mitochondria exhibit distinct morphological and biochemical properties; however the functional differences are not well understood. This study investigated the effect of fed (FED) versus 48 h total foodrestriction (FR) on rat red gastrocnemius muscle PDK2 and 4 isoform content in SS and IMF mitochondria. PDK4 content was ~3-5 fold higher in SS mitochondria compared to IMF (p=0.001), and increased with FR -3-4- fold in both subpopulations (p<0.001). PDK2 was -2.5-4 fold higher in SS mitochondria compared to IMF (p=0.001), but PDK2 was unaltered with FR. Citrate synthase activity (|imol/min/mg mitochondrial protein) was not different between either subpopulation. As well there were no significant differences between mitochondrial subpopulations in PDH complex components in both fed and FR states. These results demonstrate that there is a markedly higher content of both PDK isofonns in SS compared to IMF mitochondria. Although PDK2 does not increase in either subpopulation in response to FR, PDK4 increases to a similar extent in both SS and IMF after 48 h food-restriction.

Preliminary epidemiological study of latent tuberculosis in Mexican agricultural workers in the Niagara region, Canada

It is well documented that the majority of Tuberculosis (TB) cases diagnosed in Canada are related to foreign-bom persons from TB high-burden countries. The Canadian seasonal agricultural workers program (SAWP) operating with Mexico allows migrant workers to enter the country with a temporary work permit for up to 8 months. Preiimnigration screening of these workers by both clinical examination and chest X-ray (CXR) reduces the risk of introducing cases of active pulmonary TB to Canada, but screening for latent TB (LTBI) is not routinely done. Studies carried out in industrialized nations with high immigration from TBendemic countries provide data of lifetime LTBI reactivation of around 10% but little is known about reactivation rates within TB-endemic countries where new infections (or reinfections) may be impossible to distinguish from reactivation. Migrant populations like the SAWP workers who spend considerable amounts of time in both Canada and TBendemic rural areas in Mexico are a unique population in terms of TB epidemiology. However, to our knowledge no studies have been undertaken to explore either the existence of LTBI among Mexican workers, the probability of reactivation or the workers' exposure to TB cases while back in their communities before returning the following season. Being aware of their LTBI status may help workers to exercise healthy behaviours to avoid TB reactivation and therefore continue to access the SAWP. In order to assess the prevalence of LTBI and associated risk factors among Mexican migrant workers a preliminary cross sectional study was designed to involve a convenience sample of the Niagara Region's Mexican workers in 2007. Research ethics clearance was granted by Brock University. Individual questionnaires were administered to collect socio-demographic and TB-related epidemiological data as well as TB knowledge and awareness levels. Cellular immunity to M tuberculosis was assessed by both an Interferon-y release assay (lGRA), QuantiFERON -TB Gold In-Tube (QFf™) and by the tuberculin skin test (TSn using Mantoux. A total of 82 Mexican workers (out of 125 invited) completed the study. Most participants were male (80%) and their age ranged from 22 to 65 years (mean 38.5). The prevalence of LTBI was 34% using TST and 18% using QFTTM. As previously reported, TST (using ~lOmm cut-off) showed a sensitivity of 93.3% and a specificity of 79.1 %. These findings at the moment cannot predict the probability of progression to active TB; only longitudinal cohort studies of this population can ascertain this outcome. However, based on recent publications, lORA positive individuals may have up to 14% probability of reactivation within the next two years. Although according to the SA WP guidelines, all workers undergo TB screening before entering or re-entering Canada, CXR examination requirements showed to be inconsistent for this population: whereas 100% of the workers coming to Canada for the first time reported having the procedure done, only 31 % of returning participants reported having had a CXR in the past year. None of the participants reported ever having a CXR compatible with TB which was consistent with the fact that none had ever been diagnosed with active pulmonary TB and with only 3.6% reporting close contact with a person with active TB in their lifetime. Although Mexico reports that 99% of popUlation is fully immunized against TB within the first year of age, only 85.3% of participants reported receiving BOC vaccine in childhood. Conversely, even when TST is not part of the routine TB screening in endemic countries, a suqDrisingly high 25.6% reported receiving a TST in the past. In regards to TB knowledge and awareness, 74% of the studied population had previous knowledge about (active) TB, 42% correctly identified active TB symptomatology, 4.8% identified the correct route of transmission, 4.8% knew about the existence of LTBI, 3.6% knew that latent TB could reactivate and 48% recognized TB as treatable and curable. Of all variables explored as potential risk factors for LTBI, age was the only one which showed statistical significance. Significant associations could not be proven for other known variables (such as sex, TB contact, history of TB) probably because of the small sample size and the homogeneity of the sample. Screening for LTBI by TST (high sensitivity) followed by confirmation with QFT''"'^ (high specificity) suggests to be a good strategy especially for immigrants from TB high-burden countries. After educational sessions, workers positive for LTBI gained greater knowledge about the signs and symptoms of TB reactivation as well as the risk factors commonly associated with reactivation. Additionally, they were more likely to attend their annual health check up and request a CXR exam to monitor for TB reactivation.

Carbohydrate refeeding rapidly reverses the adaptive upregulation of human skeletal muscle pyruvate dehydrogenase kinase following a high fat diet

The time course for the reversal of the adaptive increase in pyruvate dehydrogenase kinase (PDK) activity following a 6d high fat diet (HP: 4.2 ± 0.2 % carbohydrate; 75.6 ± 0.4 % fat; 19.5 ± 0.8 % protein) was investigated in human skeletal muscle (vastus lateralis). HF feeding increased PDK activity by 44% (from 0.081 ± 0.025 min"' to 0.247 ± 0.025 mm\p < 0.05). Following carbohydrate re-feeding, (88% carbohydrate; 5% fat; 7% protein), PDK activity had returned to baseline (0.111 ± 0.014 min"') within 3h of re-feeding. The active fraction of pyruvate dehydrognease (PDHa) was depressed following 6d of the HF diet (from 0.89 ± 0.21 mmol/min/kg WW to 0.32 ± 0.05 mmol/min/kg ww,p <0.05) and increased to pre-HF levels by 45 min of post re-feeding (0.74 ±0.19 mmol/min/kg ww) and remained elevated for 3h. Western blotting analysis of the PDK isoforms, PDK4 and PDK2, revealed a 31% increase in PDK4 protein content following the HF diet, with no change in PDK2 protein. This adaptive increase in PDK4 protein content was reversed with carbohydrate re-feeding. It was concluded that the adaptive up-regulation in PDK activity and PDK4 protein content was fiilly reversed by 3h following carbohydrate re-feeding.

Pyruvate dehydrogenase activity in response to skeletal muscle contraction at two stimulation frequencies in pyruvate dehydrogenase kinase 2 knockout mice

Pyruvate dehydrogenase (PDH) plays an important role in regulating carbohydrate oxidation in skeletal muscle. PD H is deactivated by a set of PD H kinases (PD K 1-4) with PDK2 and 4 being the predominant isoforms in skeletal muscle. PDK2 is highly sensitive to pyruvate inhibition, and is the most abundant isoform, while PDKI and 4 protein content are normally lower. This study examined the PDK isoform content and PDHa activation in muscle at rest and 10 and 40 Hz stimulation from PDK2 knockout (PDK2KO) mice to delineate the role of PDK2 in activating the PDH complex during low and moderate intensity muscle contraction. PDHa activity was lower in PDK2KO mice during contraction while total PDK actitvity was -4 fold lower. PDK4 protein was not different, however PDKI partially compensated for the lack of PDK2 and was -56% higher than WT. PDKI is a very potent inhibitor of the PDH complex due to its phosphorylation site specificity and allosteric regulation. These results suggest that the site specificity and allosteric regulatory properties of the individual PDK isoforms are more important than total PDK activity in determining transformation of the complex and PDHa activity during acute muscle contraction.

Vitamin D status and latent tuberculosis infection : a preliminary study in a group of healthy Mexican agricultural workers

Vitamin D metabolites are important in the regulation of bone and calcium homeostasis, but also have a more ubiquitous role in the regulation of cell differentiation and immune function. Severely low circulating 25-dihydroxyvitamin D [25(OH)D] concentrations have been associated with the onset of active tuberculosis (TB) in immigrant populations, although the association with latent TB infection (LTBI) has not received much attention. A previous study identified the prevalence of LTBI among a sample of Mexican migrant workers enrolled in Canada's Seasonal Agricultural Workers Program (SA WP) in the Niagara Region of Ontario. The aim of the present study was to determine the vitamin D status of the same sample, and identify if a relationship existed with LTBI. Studies of vitamin D deficiency and active TB are most commonly carried out among immigrant populations to non-endemic regions, in which reactivation of LTBI has occurred. Currently, there is limited knowledge of the association between vitamin D deficiency and LTBI. Entry into Canada ensured that these individuals did not have active TB, and L TBI status was established previously by an interferon-gamma release assay (IGRA) (QuantiFERON-TB Gold In-Tube®, Cellestis Ltd., Australia). Awareness of vitamin D status may enable individuals at risk of deficiency to improve their nutritional health, and those with LTBI to be aware of this risk factor for disease. Prevalence of vitamin D insufficiency among the Mexican migrant workers was determined from serum samples collected in the summer of 2007 as part of the cross sectional LTBI study. Samples were measured for concentrations of the main circulating vitamin D metabolite, 25(OH)D, with a widely used 1251 250HD RIA (DiaSorin Inc.®, Stillwater, MN), and were categorized as deficient «37.5 nmoI/L), insufficient (>37.5 nmollL, < 80 nmol/L) or sufficient (2::80 nmoI/L). Fisher's exact tests and t tests were used to determine if vitamin D status (sufficiency or insufficiency) or 25(OH)D concentrations significantly differed by sex or age categories. Predictors of vitamin D insufficiency and 25(OH)D concentrations were taken from questionnaires carried out during the previous study, and analyzed in the present study using multiple regression prediction models. Fisher's exact test and t test was used to determine if vitamin D status or 25(OH)D concentration differed by LTBI status. Strength of the relationship between interferongamma (IFN-y) concentration (released by peripheral T cells in response to TB antigens) and 25(OH)D concentration was analyzed using a Spearman correlation. Out of 87 participants included in the study (78% male; mean age 38 years), 14 were identified as LTBI positive but none had any signs or symptoms of TB reactivation. Only 30% of the participants were vitamin D sufficient, whereas 68% were insufficient and 2% were deficient. Significant independent predictors of lower 25(OH)D concentrations were sex, number of years enrolled in the SA WP and length of stay in Canada. No significant differences were found between 25(OH)D concentrations and LTBI status. There was a significant moderate correlation between IFN-y and 25(OH)D concentrations ofLTBI-positive individuals. The majority of participants presented with Vitamin D insufficiency but none were severely deficient, indicating that 25(OH)D concentrations do not decrease dramatically in populations who temporarily reside in Canada but go back to their countries of origin during the Canadian winter. This study did not find a statistical relationship between low levels of vitamin D and LTBI which suggests that in the presence of overall good health, lower than ideal levels of 2S(OH)D, may still be exerting a protective immunological effect against LTBI reactivation. The challenge remains to determine a critical 2S(OH)D concentration at which reactivation is more likely to occur.

The Role of Pyruvate Dehydrogenase Kinase-4 in Post-Exercise Glycogen Recovery

The pyruvate dehydrogenase (PDH) complex regulates the oxidation of carbohydrates in mammals. Decreased activation of PDH following exhaustive exercise may aid the resynthesis of glycogen through increased activity of PDH kinase-4 (PDK4), one of four kinases that decrease the activity of the PDH complex. The purpose of this study was to examine the role of PDK4 in post-exercise glycogen resynthesis. Wild-type (WT) and PDK4-knockout (PDK4-KO mice) were exercised to exhaustion and were sampled at rest (Rest), at exercise exhaustion (Exh), and after two-hours post-exercise (Rec). Differences in feeding post-exercise led to the addition of a PDK4-KO group, pair-fed (PF) with WT mice. Glycogen fully recovered in all Rec groups in muscle however remained low in the PF group in liver. Flux through PDH was elevated in PDK4-KO muscle with feeding and low in the PF group in both tissues. This suggests PDK4 may fine-tune flux through PDH during exercise recovery.