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Nutrients affect adult stem cells through complex mechanisms involving multiple organs. Adipocytes are highly sensitive to diet and have key metabolic roles, and obesity increases the risk for many cancers. How diet-regulated adipocyte metabolic pathways influence normal stem cell lineages, however, remains unclear. has highly conserved adipocyte metabolism and a well-characterized female germline stem cell (GSC) lineage response to diet. Here, we conducted an isobaric tags for relative and absolute quantification (iTRAQ) proteomic analysis to identify diet-regulated adipocyte metabolic pathways that control the female GSC lineage. On a rich (relative to poor) diet, adipocyte Hexokinase-C and metabolic enzymes involved in pyruvate/acetyl-CoA production are upregulated, promoting a shift of glucose metabolism toward macromolecule biosynthesis. Adipocyte-specific knockdown shows that these enzymes support early GSC progeny survival. Further, enzymes catalyzing fatty acid oxidation and phosphatidylethanolamine synthesis in adipocytes promote GSC maintenance, whereas lipid and iron transport from adipocytes controls vitellogenesis and GSC number, respectively. These results show a functional relationship between specific metabolic pathways in adipocytes and distinct processes in the GSC lineage, suggesting the adipocyte metabolism-stem cell link as an important area of investigation in other stem cell systems.
Copyright © 2017 by the Genetics Society of America.
Oxidative phosphorylation (OXPHOS) efficiency, defined as the ATP-to-O ratio, is a critical feature of mitochondrial function that has been implicated in health, aging, and disease. To date, however, the methods to measure ATP/O have primarily relied on indirect approaches or entail parallel rather than simultaneous determination of ATP synthesis and O2 consumption rates. The purpose of this project was to develop and validate an approach to determine the ATP/O ratio in permeabilized fiber bundles (PmFBs) from simultaneous measures of ATP synthesis (JATP) and O2 consumption (JO2 ) rates in real time using a custom-designed apparatus. JO2 was measured via a polarigraphic oxygen sensor and JATP via fluorescence using an enzyme-linked assay system (hexokinase II, glucose-6-phosphate dehydrogenase) linked to NADPH production. Within the dynamic linear range of the assay system, ADP-stimulated increases in steady-state JATP mirrored increases in steady-state JO2 (r(2) = 0.91, P < 0.0001, n = 57 data points). ATP/O ratio was less than one under low rates of respiration (15 μM ADP) but increased to more than two at moderate (200 μM ADP) and maximal (2,000 μM ADP) rates of respiration with an interassay coefficient of variation of 24.03, 16.72, and 11.99%, respectively. Absolute and relative (to mechanistic) ATP/O ratios were lower in PmFBs (2.09 ± 0.251, 84%) compared with isolated mitochondria (2.44 ± 0.124, 98%). ATP/O ratios in PmFBs were not affected by the activity of adenylate kinase or creatine kinase. These findings validate an enzyme-linked respiratory clamp system for measuring OXPHOS efficiency in PmFBs and provide evidence that OXPHOS efficiency increases as energy demand increases.
Copyright © 2016 the American Physiological Society.
Protein hyperacetylation is associated with glucose intolerance and insulin resistance, suggesting that the enzymes regulating the acetylome play a role in this pathological process. Sirtuin 3 (SIRT3), the primary mitochondrial deacetylase, has been linked to energy homeostasis. Thus, it is hypothesized that the dysregulation of the mitochondrial acetylation state, via genetic deletion of SIRT3, will amplify the deleterious effects of a high-fat diet (HFD). Hyperinsulinemic-euglycemic clamp experiments show, for the first time, that mice lacking SIRT3 exhibit increased insulin resistance due to defects in skeletal muscle glucose uptake. Permeabilized muscle fibers from HFD-fed SIRT3 knockout (KO) mice showed that tricarboxylic acid cycle substrate-based respiration is decreased while fatty acid-based respiration is increased, reflecting a fuel switch from glucose to fatty acids. Consistent with reduced muscle glucose uptake, hexokinase II (HKII) binding to the mitochondria is decreased in muscle from HFD-fed SIRT3 KO mice, suggesting decreased HKII activity. These results show that the absence of SIRT3 in HFD-fed mice causes profound impairments in insulin-stimulated muscle glucose uptake, creating an increased reliance on fatty acids. Insulin action was not impaired in the lean SIRT3 KO mice. This suggests that SIRT3 protects against dietary insulin resistance by facilitating glucose disposal and mitochondrial function.
© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.
AMP-activated protein kinase (AMPK) has been identified as a regulator of gene transcription, increasing mitochondrial proteins of oxidative metabolism as well as hexokinase expression in skeletal muscle. In mice, muscle-specific knockout of LKB1, a component of the upstream kinase of AMPK, prevents contraction- and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR)-induced activation of AMPK in skeletal muscle, and the increase in hexokinase II protein that is normally observed with chronic AICAR activation of AMPK. Since previous reports show a cAMP response element in the promoter region of the hexokinase II gene, we hypothesized that the cAMP-response element (CRE) binding protein (CREB) family of transcription factors could be targets of AMPK. Using radioisotopic kinase assays, we found that recombinant and rat liver and muscle AMPK phosphorylated CREB1 at the same site as cAMP-dependent protein kinase (PKA). AMPK was also found to phosphorylate activating transcription factor 1 (ATF1), CRE modulator (CREM), and CREB-like 2 (CREBL2), but not ATF2. Treatment of HEK-293 cells stably transfected with a CREB-driven luciferase reporter with AICAR increased luciferase activity approximately threefold over a 24-h time course. This increase was blocked with compound C, an AMPK inhibitor. In addition, AICAR-induced activation of AMPK in incubated rat epitrochlearis muscles resulted in an increase in both phospho-acetyl-CoA carboxylase and phospho-CREB. We conclude that CREB and related proteins are direct downstream targets for AMPK and are therefore likely involved in mediating some effects of AMPK on expression of genes having a CRE in their promoters.
OBJECTIVE - Muscle glucose uptake (MGU) is regulated by glucose delivery to, transport into, and phosphorylation within muscle. The aim of this study was to determine the role of limitations in glucose phosphorylation in the control of MGU during either physiological insulin stimulation (4 mU x kg(-1) x min(-1)) or exercise with chow or high-fat feeding.
RESEARCH DESIGN AND METHODS - C57BL/6J mice with (HK(+/-)) and without (WT) a 50% hexokinase (HK) II deletion were fed chow or high-fat diets and studied at 4 months of age during a 120-min insulin clamp or 30 min of treadmill exercise (n = 8-10 mice/group). 2-deoxy[(3)H]glucose was used to measure R(g), an index of MGU.
RESULTS - Body weight and fasting arterial glucose were increased by high-fat feeding and partial HK II knockout (HK(+/-)). Both high-fat feeding and partial HK II knockout independently created fasting hyperinsulinemia, a response that was increased synergistically with combined high-fat feeding and HK II knockout. Whole-body insulin action was suppressed by approximately 25% with either high-fat feeding or partial HK II knockout alone but by >50% when the two were combined. Insulin-stimulated R(g) was modestly impaired by high-fat feeding and partial HK II knockout independently ( approximately 15-20%) but markedly reduced by the two together ( approximately 40-50%). Exercise-stimulated R(g) was reduced by approximately 50% with high-fat feeding and partial HK II knockout alone and was not attenuated further by combining the two.
CONCLUSIONS - In summary, impairments in whole-body metabolism and MGU due to high-fat feeding and partial HK II knockout combined during insulin stimulation are additive. In contrast, combining high-fat feeding and partial HK II knockout during exercise causes no greater impairment in MGU than the two manipulations independently. This suggests that MGU is impaired during exercise by high-fat feeding due to, in large part, a limitation in glucose phosphorylation. Together, these studies show that the high-fat-fed mouse is characterized by defects at multiple steps of the MGU system that are precipitated by different physiological conditions.
The absence of GLUT4 severely impairs basal glucose uptake in vivo, but does not alter glucose homeostasis or circulating insulin. Glucose uptake in isolated contracting skeletal muscle (MGU) is also impaired by the absence of GLUT4, and onset of muscle fatigue is hastened. Whether the body can compensate and preserve glucose homeostasis during exercise, as it does in the basal state, is unknown. One aim was to test the effectiveness of glucoregulatory compensation for the absence of GLUT4 in vivo. The absence of GLUT4 was also used to further define the role of hexokinase (HK) II, which catalyses glucose phosphorylation after it is transported in the cell. HK II increases MGU during exercise, as well as exercise endurance. In the absence of GLUT4, HK II expression will not affect MGU. A second aim was to test whether, in the absence of GLUT4, HK II retains its ability to increase exercise endurance. Wild-type (WT), GLUT4 null (GLUT4(-/-)), and GLUT4 null overexpressing HK II (GLUT4(-/-)HK(Tg)) mice were studied using a catheterized mouse model that allows blood sampling and isotope infusions during treadmill exercise. The impaired capacity of working muscle to take up glucose in GLUT4(-/-) is partially offset by an exaggerated increase in the glucagon: insulin ratio, increased liver glucose production, hyperglycaemia, and a greater capillary density in order to increase the delivery of glucose to the exercising muscle of GLUT4(-/-). Hearts of GLUT4(-/-) also exhibited a compensatory increase in HK II expression and a paradoxical increase in glucose uptake. Exercise tolerance was reduced in GLUT4(-/-) compared to WT. As expected, MGU in GLUT4(-/-)HK(Tg) was the same as in GLUT4(-/-). However, HK II overexpression retained its ability to increase exercise endurance. In conclusion, unlike the basal state where glucose homeostasis is preserved, hyperglycaemia results during exercise in GLUT4(-/-) due to a robust stimulation of liver glucose release in the face of severe impairments in MGU. Finally, studies in GLUT4(-/-)HK(Tg) show that HK II improves exercise tolerance, independent of its effects on MGU.
Previous studies in mice suggest that portal venous infusion of glucose at a low rate paradoxically causes hypoglycemia; this does not occur in dogs, rats, and humans. A possible explanation is that fasting status in the mouse studies may have altered the response. We sought to determine whether the response to portal glucose delivery in the mouse was similar to that seen in other species and whether it was dependent on fasting status. Studies were performed on chronically catheterized conscious mice. Catheters were placed into the portal and jugular veins and carotid artery 5 days before study. After a 5- or 16-h fast, glucose was infused into either the portal (PO) or the jugular vein (JU) for 6 h at 25 microg.g(-1).min(-1). [3-(3)H]glucose was infused into the JU to measure glucose turnover. In 5-h-fasted mice, PO and JU exhibited similar increases in arterial blood glucose from 155 +/- 11 to 173 +/- 19 and 147 +/- 8 to 173 +/- 10 mg/dl, respectively. Endogenous glucose production decreased and arterial insulin increased to the same extent in both PO and JU. A similar response was observed in 16-h-fasted mice; however, the proportion of hepatic glycogen synthesis occurring by the indirect pathway was increased by fasting. In summary, portal glucose delivery in the mouse did not cause hypoglycemia even when the duration of the fast was extended. The explanation of the differing response from previous reports in the mouse is unclear.
Using an expression cloning strategy, we have identified TFE3, a basic helix-loop-helix protein, as a transactivator of metabolic genes that are regulated through an E-box in their promoters. Adenovirus-mediated expression of TFE3 in hepatocytes in culture and in vivo strongly activated expression of IRS-2 and Akt and enhanced phosphorylation of insulin-signaling kinases such as Akt, glycogen synthase kinase 3beta and p70S6 kinase. TFE3 also induced hexokinase II (HK2) and insulin-induced gene 1 (INSIG1). These changes led to metabolic consequences, such as activation of glycogen and protein synthesis, but not lipogenesis, in liver. Collectively, plasma glucose levels were markedly reduced both in normal mice and in different mouse models of diabetes, including streptozotocin-treated, db/db and KK mice. Promoter analyses showed that IRS2, HK2 and INSIG1 are direct targets of TFE3. Activation of insulin signals in both insulin depletion and resistance suggests that TFE3 could be a therapeutic target for diabetes.
Hexokinase (HK) II content is elevated in fatigue resistant muscle fibres and exercise trained muscle. The aim of this study was to determine if exercise capacity is dependent on muscle HK protein content. C57Bl/6 mice with a 50% HK knockout (HK+/-), no genetic manipulation (wild-type, WT) and an approximately 3-fold HK overexpression (HKTg) were tested. Mice (n = 12/group) completed both a maximal oxygen consumption test(VO2max) test and an endurance capacity test (run at approximately 75% VO2max) on an enclosed treadmill equipped to measure gas exchange. Arterial and venous catheters were surgically implanted into separate groups of mice (n = 9-11/group) in order to measure an index of muscle glucose uptake Rg during 30 min of treadmill exercise. Maximum work rate (0.95 +/- 0.05, 1.00 +/- 0.04 and 1.06 +/- 0.07 kg m min-1), (137 +/- 3, 141 +/- 4 and 141 +/- 5 ml kg-1 min-1) and maximal respiratory exchange ratio (1.04 +/- 0.02, 1.00 +/- 0.03 and 1.04 +/- 0.04) were similar in HK+/-, WT and HKTg, respectively. Exercise endurance capacity (measured as time to exhaustion) increased as HK content increased (55 +/- 11, 77 +/- 5 and 98 +/- 9 min) and this was related to Rg measured in mice during 30 min of exercise (13 +/- 2, 24 +/- 5 and 42 +/- 5 micromol (100 g)-1 min-1). Muscle glycogen in sedentary HK+/-mice and HK+/- mice following 30 min of exercise were significantly lower than in HKTg and WT mice. However, the net exercise-induced muscle glycogen breakdown was equal in the three genotypes. In summary, HK protein content within the range studied (a) was not associated with a difference in the capacity to perform maximal intensity exercise, (b) was a powerful determinant of the ability to sustain moderate intensity exercise, as reducing HK content impaired endurance and increasing HK content enhanced endurance, and (c) although directly related to exercise endurance, was not a determinant of net muscle glycogen usage during exercise. In conclusion, adaptations that increase HK protein content and/or functional activity such as regular exercise contribute to increased muscular endurance.
The aim of this study was to test whether in fact glucose transport is rate-limiting in control of muscle glucose uptake (MGU) under physiological hyperinsulinaemic conditions in the conscious, unrestrained mouse. C57Bl/6J mice overexpressing GLUT4 (GLUT4(Tg)), hexokinase II (HK(Tg)), or both (GLUT4(Tg) + HK(Tg)), were compared to wild-type (WT) littermates. Catheters were implanted into a carotid artery and jugular vein for sampling and infusions at 4 month of age. After a 5-day recovery period, conscious mice underwent one of two protocols (n = 8-14/group) after a 5-h fast. Saline or insulin (4 mU kg(-1) min(-1)) was infused for 120 min. All mice received a bolus of 2-deoxy[(3)H]glucose (2-(3)HDG) at 95 min. Glucose was clamped at approximately 165 mg dl(-1) during insulin infusion and insulin levels reached approximately 80 microU ml(-1). The rate of disappearance of 2-(3)HDG from the blood provided an index of whole body glucose clearance. Gastrocnemius, superficial vastus lateralis and soleus muscles were excised at 120 min to determine 2-(3)HDG-6-phosphate levels and calculate an index of MGU (R(g)). Results show that whole body and tissue-specific indices of glucose utilization were: (1) augmented by GLUT4 overexpression, but not HKII overexpression, in the basal state; (2) enhanced by HKII overexpression in the presence of physiological hyperinsulinaemia; and (3) largely unaffected by GLUT4 overexpression during insulin clamps whether alone or combined with HKII overexpression. Therefore, while glucose transport is the primary barrier to MGU under basal conditions, glucose phosphorylation becomes a more important barrier during physiological hyperinsulinaemia in all muscles. The control of MGU is distributed rather than confined to a single rate-limiting step such as glucose transport as glucose transport and phosphorylation can both become barriers to skeletal muscle glucose influx.