Differential Metabolic Regulation Research Articles

The abundance of the Calvin–Benson proteoforms is not affected by growth at CO2-enriched atmosphere in Eucalyptus young plants

Mitigation of climate change is an uttermost necessity and plants play an important role in removing atmospheric CO2. Besides being the primary source for the production of pulp and paper, fast-growing evergreen trees have a huge potential to assimilate and stock massive amounts of atmospheric carbon. Previous proteomics study indicated that growth of Eucalyptus urophylla plants in an ultra-high CO2 atmosphere (i.e. 1,000 ppm) increased the abundance of carbon assimilation enzyme proteins. Here, we used proteomics as a multidimensional technology to understand the metabolic changes that occur in Eucalyptus leaves when young plants were cultivated in a higher than current CO2 levels (i.e. 680 ppm). After 30 days of treatment, plant species that presented the most dissimilar phenotypic responses were E. pellita, E. urophylla and E. grandis grown at 680 ppm of CO2. Proteome investigation indicated the absence of a consistent increase in the abundance of the proteoforms and enzymes involved in carbon assimilation. Large-scale multivariate analysis and gene ontology term enrichment suggested that CO2, at the aforementioned concentration, increased the number of photosystem-related proteins. Unique proteome features were identified in the Eucalyptus species when stimulated by CO2. Our data refutes the previous findings on the increase in the number of carbon assimilation proteins upon CO2 cultivation and provides new insights into a differential metabolic regulation in Eucalyptus plants according to the CO2 levels plants are grown in. Mass spectrometric data are available via ProteomeXchange under the identifier PXD044997.

SAT650 Catecholamine-independent Neural Pathways Drive The Rapid Catabolism Of Metabolically Inert Fat

Abstract Disclosure: X. Zhang: None. A. Majumdar: None. B. Kleiboeker: None. K.L. Magee: None. B.S. Learman: None. S.A. Thomas: None. I.J. Lodhi: None. O.A. MacDougald: None. E.L. Scheller: None. Adipocytes classically store or release energy in response to changes in metabolic status. However, several fat depots throughout the body are resistant to these metabolic cues. Identification of novel mechanisms that drive energy release from metabolically inert adipocytes is needed to inform strategies to deplete stubborn fat and, conversely, to preserve these depots in pathologic settings of wasting and cachexia. To address this question, we focused on the constitutive bone marrow adipose tissue (cBMAT), a well-established site of metabolically inert fat that remains unchanged with fasting, exercise, and cold exposure and is depleted only in certain extreme conditions such as cachexia or starvation. To study this unique depot, we developed a mouse model of rapid, complete depletion of all fat, including cBMAT, within 9 days using central intracerebroventricular (ICV) injection of leptin. Surgical denervation, chemical sympathectomy, or genetic dopamine beta-hydroxylase (DBH) deletion did not prevent the ICV leptin-induced depletion of cBMAT, revealing this process to be independent of catecholamines and the sympathetic nervous system (SNS). Vossicle implantation defined the mediators as transported through the circulation, and BMAT-specific ablation of adipose triglyceride lipase (ATGL) identified dependence on basal lipolysis. Gene expression and a radioisotope-based de novo lipogenesis assay pinpointed a concurrent suppression of lipogenesis, and subcutaneous insulin supplementation prevented leptin-induced cBMAT depletion, but not other depots. Together, this defines the differential regulation of metabolism in metabolically inert fat depots such as cBMAT. Specifically, leptin-responsive circuits in the brain can deplete inert adipocytes by suppressing lipogenesis while maintaining basal lipolysis independent of the SNS. Presentation: Saturday, June 17, 2023

The crisscross between p53 and metabolism in cancer.

As the guardian of the genome, p53 is well known for its tumor suppressor function in humans, controlling cell proliferation, senescence, DNA repair and cell death in cancer through transcriptional and non-transcriptional activities. p53 is the most frequently mutated gene in human cancer, but how its mutation or depletion leads to tumorigenesis still remains poorly understood. Recently, there has been increasing evidence that p53 plays a vital role in regulating cellular metabolism as well as in metabolic adaptation to nutrient starvation. In contrast, mutant p53 proteins, especially those harboring missense mutations, have completely different functions compared to wild-type p53. In this review, we briefly summarize what is known about p53 mediating anabolic and catabolic metabolism in cancer, and in particular discuss recent findings describing how metabolites regulate p53 functions. To illustrate the variability and complexity of p53 function in metabolism, we will also review the differential regulation of metabolism by wild-type and mutant p53.

Metabolome-Wide Reprogramming Modulated by Wnt/β-Catenin Signaling Pathway.

A family of signal transduction pathways known as wingless type (Wnt) signaling pathways is essential to developmental processes like cell division and proliferation. Mutation in Wnt signaling results in a variety of diseases, including cancers of the breast, colon, and skin, metabolic disease, and neurodegenerative disease; thus, the Wnt signaling pathways have been attractive targets for disease treatment. However, the complicatedness and large involveness of the pathway often hampers pinpointing the specific targets of the metabolic process. In our current study, we investigated the differential metabolic regulation by the overexpression of the Wnt signaling pathway in a timely-resolved manner by applying high-throughput and un-targeted metabolite profiling. We have detected and annotated 321 metabolite peaks from a total of 36 human embryonic kidney (HEK) 293 cells using GC-TOF MS and LC-Orbitrap MS. The un-targeted metabolomic analysis identified the radical reprogramming of a range of central carbon/nitrogen metabolism pathways, including glycolysis, TCA cycle, and glutaminolysis, and fatty acid pathways. The investigation, combined with targeted mRNA profiles, elucidated an explicit understanding of activated fatty acid metabolism (β-oxidation and biosynthesis). The findings proposed detailed mechanistic biochemical dynamics in response to Wnt-driven metabolic changes, which may help design precise therapeutic targets for Wnt-related diseases.

Prevalence of Acquired N-Glycosylation Sites at the Single Cell Level in Follicular Lymphoma

Follicular lymphoma (FL), a malignancy derived from germinal center B cells, is characterized by ongoing somatic hypermutation of the immunoglobulin variable region (IGV) genes. This somatic hypermutation can introduce novel N-glycosylation sites (N-sites), consisting of Asn-X-Ser/Thr motifs, in the IGV resulting in the addition of glycans at these sites. Binding of lectins, such as DC-SIGN, to these glycosylated surface immunoglobulins (i.e., B cell receptors) activates the B cell receptor pathway in an antigen-independent manner, promoting FL B cell survival and proliferation. Previous studies have concluded that the acquisition of N-sites is a stable and clonal event during FL evolution (Odabashian et al., Blood 2020). Here, we investigated the dynamics and potential impact of N-site prevalence at a single cell level, in paired heavy and light chains, across sites and over time in 16 patients with FL. We profiled tumor fine needle aspirates from patients enrolled in two clinical trials (NCT02927964, NCT03410901). 15 of 16 patients were previously, but not recently treated, while one was previously untreated. Samples were collected from different tumor sites before treatment, on-treatment, and at tumor progression. Single cell suspensions were subjected to single cell B cell receptor (scBCR) and RNA (scRNA) sequencing using the 10X chromium platform. Only BCR sequences belonging to patient-specific dominant malignant heavy and light chain clones were considered for further analysis. In total, we analyzed more than 250,000 FL tumor cells from 89 tumor samples from 16 FL patients. While most patients acquired at least one N-site as an early and stable event that was conserved across distinct tumor sites and over time, 3 patient cases proved to be exceptions: one patient had no detectable N-sites in either the heavy or light chain IGV genes at any tumor site or timepoint. In a second patient, only one of two tumor sites exhibited a novel N-site, and this was probably acquired after divergence of the tumor populations to distinct tumor sites (Haebe, Shree et al., Blood 2021). Interestingly, clinical tumor progression in this patient arose from selection and expansion of an N-site negative subclone, despite the putative survival advantage conferred by acquired N-sites. In the third patient, N-sites were acquired in a precursor cell before subclonal divergence, but subsequently overtaken by an N-site-negative subpopulation, which comprised up to 90% of tumor cells at a given site (Figure 1A). These results suggest that other acquired features can out-compete the selective advantage of N-glycosylation. To elucidate biological differences between cells with and without N-sites within each sample, we integrated scBCR and scRNA data for the third patient using shared cell barcodes. N-site positive cells exhibited a higher expression of the BCR pathway and inflammatory response, while cells without N-sites had higher activity of metabolic processes, including oxidative phosphorylation and glycolysis (Figure 1B). In conclusion, while acquired N-sites likely support FL pathogenesis through antigen-independent BCR signaling in many FL patients, selection and expansion of N-site negative cells in some patients in our study suggests that N-site negative cells can thrive, and likely depend on other pathways, with our preliminary results pointing to differential metabolic regulation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Increased Heat Resilience of Intraspecific Outbred Compared to Inbred Lineages in the Kelp Laminaria digitata: Physiology and Transcriptomics

Marine forests and kelps as their foundation species are threatened by ocean warming especially at the warm distributional edges. Previously identified genetic divergence and ecotypic differentiation within kelp species may allow to produce more resilient lineages by intraspecific outbreeding among populations. In a mechanistic investigation of heat stress, heterosis (hybrid vigour), and underlying gene expression patterns, we assessed the thermal performance of inbred (selfings) and outbred (reciprocal crosses) sporophytes of the N-Atlantic kelp Laminaria digitata among clonal isolates from two divergent populations; one from the temperate North Sea (Helgoland) and one from the Arctic (Spitsbergen). First, we investigated the upper thermal tolerance of microscopic sporophytes in a 14-day experiment applying sublethal to lethal 20–23°C. The upper survival temperature of microscopic sporophytes was lower for the inbred Arctic selfing (21°C) than for the temperate selfing and the reciprocal crosses (22°C). Only in the temperate selfing, 4.5% of sporophytes survived 23°C. We then subjected 4–7 cm long sporophytes to a control temperature (10°C), moderate (19°C) and sublethal to lethal heat stress (20.5°C) for 18 days to assess gene expression in addition to physiological parameters. Growth and optimum quantum yield decreased similarly in the reciprocal crosses and the temperate selfing at 19 and 20.5°C, while inbred Arctic sporophytes died within seven days at both 19 and 20.5°C. In response to 20.5°C, 252 genes were constitutively regulated across all surviving lineages, which we use to describe metabolic regulation patterns in response to heat stress in kelp. At sublethal 20.5°C, ca. 150 genes were differentially expressed by either crossed lineage in comparison to the temperate selfing, indicating that they maintained a growth response similar to the temperate selfing with differential metabolic regulation during sublethal heat stress. Subtle differences in physiology and the differential expression of nine genes between the reciprocal crosses at 20.5°C indicate that female and male gametophytes may contribute differently to offspring traits. We consider potential inbreeding depression in the Spitsbergen selfing and quantify the better performance of both crosses using heterosis-related parameters. We discuss the potential and risks of outbreeding to produce more resilient crops for mariculture and marine forest restoration.

Metabolism as a guiding force for immunity.

Recent studies indicate that cellular metabolism plays a key role in supporting immune cell maintenance and development. Here, we review how metabolism guides immune cell activation and differentiation to distinct cellular states, and how differential regulation of metabolism allows for context-dependent support during activation and lineage commitment. We discuss emerging principles of metabolic support of immune cell function in physiology and disease, as well as their general relevance to the field of cell biology.

Metabolomic comparison between wild Ophiocordyceps sinensis and artificial cultured Cordyceps militaris.

A systematic study on the metabolome differences between wild Ophiocordyceps sinensis and artificial cultured Cordyceps militaris was conducted using liquid chromatography-mass spectrometry. Principal component analysis and orthogonal projection on latent structure-discriminant analysis results showed that C. militaris grown on solid rice medium (R-CM) and C. militaris grown on tussah pupa (T-CM) evidently separated and individually separated from wild O. sinensis, indicating metabolome difference among wild O. sinensis, R-CM and T-CM. The metabolome differences between R-CM and T-CM indicated that C. militaris could accommodate to culture medium by differential metabolic regulation. Hierarchical clustering analysis was further performed to cluster the differential metabolites and samples based on their metabolic similarity. The higher content of amino acids (pyroglutamic acid, glutamic acid, histidine, phenylalanine and arginine), unsaturated fatty acid (linolenic acid and linoleic acid), peptides, mannitol, adenosine and succinoadenosine in O. sinensis make it as an excellent choice as a traditional Chinese medicine for invigoration or nutritional supplementation. Similar compositions with O. sinensis and easy cultivation make artificially cultured C. militaris a possible alternative to O. sinensis.

Identification of Discriminating Metabolic Pathways and Metabolites in Human PBMCs Stimulated by Various Pathogenic Agents.

Immunity and cellular metabolism are tightly interconnected but it is not clear whether different pathogens elicit specific metabolic responses. To address this issue, we studied differential metabolic regulation in peripheral blood mononuclear cells (PBMCs) of healthy volunteers challenged by Candida albicans, Borrelia burgdorferi, lipopolysaccharide, and Mycobacterium tuberculosis in vitro. By integrating gene expression data of stimulated PBMCs of healthy individuals with the KEGG pathways, we identified both common and pathogen-specific regulated pathways depending on the time of incubation. At 4 h of incubation, pathogenic agents inhibited expression of genes involved in both the glycolysis and oxidative phosphorylation pathways. In contrast, at 24 h of incubation, particularly glycolysis was enhanced while genes involved in oxidative phosphorylation remained unaltered in the PBMCs. In general, differential gene expression was less pronounced at 4 h compared to 24 h of incubation. KEGG pathway analysis allowed differentiation between effects induced by Candida and bacterial stimuli. Application of genome-scale metabolic model further generated a Candida-specific set of 103 reporter metabolites (e.g., desmosterol) that might serve as biomarkers discriminating Candida-stimulated PBMCs from bacteria-stimuated PBMCs. Our analysis also identified a set of 49 metabolites that allowed discrimination between the effects of Borrelia burgdorferi, lipopolysaccharide and Mycobacterium tuberculosis. We conclude that analysis of pathogen-induced effects on PBMCs by a combination of KEGG pathways and genome-scale metabolic model provides deep insight in the metabolic changes coupled to host defense.

Melatonin Cytotoxicity Is Associated to Warburg Effect Inhibition in Ewing Sarcoma Cells.

Melatonin kills or inhibits the proliferation of different cancer cell types, and this is associated with an increase or a decrease in reactive oxygen species, respectively. Intracellular oxidants originate mainly from oxidative metabolism, and cancer cells frequently show alterations in this metabolic pathway, such as the Warburg effect (aerobic glycolysis). Thus, we hypothesized that melatonin could also regulate differentially oxidative metabolism in cells where it is cytotoxic (Ewing sarcoma cells) and in cells where it inhibits proliferation (chondrosarcoma cells). Ewing sarcoma cells but not chondrosarcoma cells showed a metabolic profile consistent with aerobic glycolysis, i.e. increased glucose uptake, LDH activity, lactate production and HIF-1α activation. Melatonin reversed Ewing sarcoma metabolic profile and this effect was associated with its cytotoxicity. The differential regulation of metabolism by melatonin could explain why the hormone is harmless for a wide spectrum of normal and only a few tumoral cells, while it kills specific tumor cell types.

Collaborative Regulation of Development but Independent Control of Metabolism by Two Epidermis-specific Transcription Factors in Caenorhabditis elegans

Cell fate specification is typically initiated by a master regulator, which is relayed by tissue-specific regulatory proteins (usually transcription factors) for further enforcement of cell identities, but how the factors are coordinated among each other to "finish up" the specification remains poorly understood. Caenorhabditis elegans epidermis specification is initiated by a master regulator, ELT-1, that activates its targets, NHR-25 and ELT-3, two epidermis-specific transcription factors that are important for development but not for initial specification of epidermis, thus providing a unique paradigm for illustrating how the tissue-specific regulatory proteins work together to enforce cell fate specification. Here we addressed the question through contrasting genome-wide in vivo binding targets between NHR-25 and ELT-3. We demonstrate that the two factors bind discrete but conserved DNA motifs, most of which remain in proximity, suggesting formation of a complex between the two. In agreement with this, gene ontology analysis of putative target genes suggested differential regulation of metabolism but coordinated control of epidermal development between the two factors, which is supported by quantitative analysis of expression of their specific or common targets in the presence or absence of either protein. Functional validation of a subset of the target genes showed both activating and inhibitory roles of NHR-25 and ELT-3 in regulating their targets. We further demonstrated differential control of specification of AB and C lineage-derived epidermis. The results allow us to assemble a comprehensive gene network underlying C. elegans epidermis development that is likely to be widely used across species and provides insights into how tissue-specific transcription factors coordinate with one another to enforce cell fate specification initiated by its master regulator.

Distinct metabolic programs in activated T cells: opportunities for selective immunomodulation

For several decades, it has been known that T-cell activation in vitro leads to increased glycolytic metabolism that fuels proliferation and effector function. Recently, this simple model has been complicated by the observation that different T-cell subsets differentially regulate fundamental metabolic pathways under the control of distinct molecular regulators. Although the majority of these data have been generated in vitro, several recent studies have documented the metabolism of T cells activated in vivo. Here, we review the recent data surrounding the differential regulation of metabolism by distinct T-cell subsets in vitro and in vivo and discuss how differential metabolic regulation might facilitate T-cell function vis-à-vis proliferation, survival, and energy production. We further discuss the important therapeutic implications of differential metabolism across T-cell subsets and review recent successes in exploiting lymphocyte metabolism to treat immune-mediated diseases.

Differential Metabolic Regulation Governed by the Rice SUB1A Gene during Submergence Stress and Identification of Alanylglycine by 1H NMR Spectroscopy

Although the genetic mechanism of submergence survival for rice varieties containing the SUB1A gene has been elucidated, the downstream metabolic effects have not yet been evaluated. In this study, the metabolomes of Oryza sativa ssp. japonica cv. M202 and cv. M202(Sub1) were profiled using (1)H NMR spectroscopy to compare the metabolic effect of submergence stress and recovery on rice in the presence or absence of SUB1A. Significant changes were observed in the NMR resonances of compounds in pathways important for carbohydrate metabolism. The presence of SUB1A in M202(Sub1) was correlated with suppression of carbohydrate metabolism in shoot tissue, consistent with the role of SUB1A in limiting starch catabolism to fuel elongation growth. The absence of SUB1A in M202 was correlated with greater consumption of sucrose stores and accumulation of amino acids that are synthesized from glycolysis intermediates and pyruvate. Under submergence conditions, alanine, a product of pyruvate metabolism, showed the largest difference between the two varieties, but elevated levels of glutamine, glutamate, leucine, isoleucine, threonine, and valine were also higher in M202 compared with the M202(Sub1) variety. The identification and characterization of alanylglycine (AlaGly) in rice is also reported. After 3 days of submergence stress, AlaGly levels decreased significantly in both genotypes but did not recover within 1 day of desubmergence with the other metabolites evaluated. The influence of SUB1A on dynamic changes in the metabolome during complete submergence provides new insights into the functional roles of a single gene in invoking a quiescence strategy that helps stabilize crop production in submergence-prone fields.

Differential regulation of metabolism by nitric oxide andS-nitrosothiols in endothelial cells

S-nitrosation of thiols in key proteins in cell signaling pathways is thought to be an important contributor to nitric oxide (NO)-dependent control of vascular (patho)physiology. Multiple metabolic enzymes are targets of both NO and S-nitrosation, including those involved in glycolysis and oxidative phosphorylation. Thus it is important to understand how these metabolic pathways are integrated by NO-dependent mechanisms. Here, we compared the effects of NO and S-nitrosation on both glycolysis and oxidative phosphorylation in bovine aortic endothelial cells using extracellular flux technology to determine common and unique points of regulation. The compound S-nitroso-L-cysteine (L-CysNO) was used to initiate intracellular S-nitrosation since it is transported into cells and results in stable S-nitrosation in vitro. Its effects were compared with the NO donor DetaNONOate (DetaNO). DetaNO treatment caused only a decrease in the reserve respiratory capacity; however, L-CysNO impaired both this parameter and basal respiration in a concentration-dependent manner. In addition, DetaNO stimulated extracellular acidification rate (ECAR), a surrogate marker of glycolysis, whereas L-CysNO stimulated ECAR at low concentrations and inhibited it at higher concentrations. Moreover, a temporal relationship between NO- and S-nitrosation-mediated effects on metabolism was identified, whereby NO caused a rapid impairment in mitochondrial function, which was eventually overwhelmed by S-nitrosation-dependent processes. Taken together, these results suggest that severe pharmacological nitrosative stress may differentially regulate metabolic pathways through both intracellular S-nitrosation and NO-dependent mechanisms. Moreover, these data provide insight into the role of NO and related compounds in vascular (patho)physiology.

Aberrant signaling responses of cells lacking syndecan1 suggest differential metabolic regulation may underlie tumor resistance of syndecan1‐null mice

Caloric restriction (CR) has been shown to inhibit tumorigenesis in a variety of animal models. CR suppresses the action of chemical carcinogens and reduces tumor formation in transgenic mouse models. Several mechanisms thought to underlie the effects of CR have been described. Levels of IGF‐1 are reduced in animals under caloric restriction, and animals show increased insulin sensitivity. CR reduces cell proliferation, enhances apoptosis, upregulates activation of AMPK (a low energy sensor) and downregulates mTOR signaling (a nutrient responsive pathway). Syndecan1 (Sdc1), a heparan sulfate proteoglycan, has been shown to be important for tumor formation. Mice lacking Sdc1 are protected from tumorigenesis in response to a chemical carcinogen and ectopic Wnt‐1 expression. To elucidate the mechanism of tumor resistance we study mammary epithelial cells (MECs). Sdc1‐null MECs in vitro show decreased population expansion in response to Wnt3A and insulin compared to wild‐type populations. Analysis of cell kinetics demonstrates that Sdc1‐null populations undergo reduced proliferation and increased apoptosis. Western blot analysis shows increased activation of AMPK in Sdc1‐null MECs. The characteristics we observe in Sdc1‐null mice and tissues suggest that metabolic alterations may be underlying the tumor resistance of Sdc1‐null mice. This research is supported by an RO1 grant and a DOD Era of Hope grant.

Efeito da temperatura na maturação ovariana e longevidade de Anastrepha fraterculus (Wied.) (Diptera: Tephritidae)

Na Região Sul do Brasil, a incidência de Anastrepha fraterculus (Wied.) flutua durante o ano, sendo que no inverno não são detectados adultos quando se utilizam métodos convencionais de coleta. Com o uso de armadilhas especiais capturaram-se adultos, ainda que, em número muito reduzido. Duas hipóteses são sugeridas para o seu aumento populacional após o inverno, nas regiões produtoras de frutíferas: a) repovoamento da região na primavera por populações circunvizinhas; e b) regulação metabólica diferencial no desenvolvimento de alguns indivíduos que conseguem sobreviver ao período crítico. A segunda hipótese foi testada no presente trabalho, analisando-se a influência de quatro temperaturas (9, 13, 20 e 25ºC) constantes sobre a maturação ovariana e sobre a longevidade das fêmeas dessa espécie. Apenas a 25 e 20ºC ocorreu amadurecimento ovariano. A expectativa de vida foi maior nas temperaturas intermediárias (20 e 13ºC) que nas temperaturas extremas (25 e 9ºC). A relação entre maturação ovariana e longevidade foi observada a 25 e 20ºC, sendo que a 25ºC o amadurecimento ovariano foi mais rápido, mas a expectativa de vida menor. Não se pode, portanto, descartar a hipótese de regulação do desenvolvimento como mecanismo populacional em A. fraterculus para repovoamento da região nos meses subseqüentes ao inverno.

Pulsatile growth hormone secretion in normal-weight and obese men: Differential metabolic regulation during energy restriction

Metabolic changes such as obesity and fasting modulate pulsatile growth hormone (GH) release in man, but the underlying mechanisms are still elusive. We studied the temporal pattern of pulsatile GH release in five normal-weight men (mean ± SD: age, 29.8 ± 4.9 years; body mass index [BMI], 24.3 ± 1.8 kg/m 2) and five obese men (age, 27.8 ± 4.8 years; BMI, 38.9 ± 4.8 kg/m 2) during their regular energy consumption and the last 24 hours of a 96-hour fasting period. GH plasma levels were determined at 10-minute intervals and glucose level was measured every 20 minutes. GH pulse analysis was performed with three different algorithms. Insulin-like growth factor-I (IGF-I), IGF-II, IGF-binding proteins (IGFBP-1, -2, and -3), and IGF-binding capacity (IGF-BC) were evaluated in samples collected at 7:00 am, 3:00 pm, and 11:00 pm. Twenty-four-hour mean GH was basally higher in normal subjects (1.1 ± 0.6 mU/L) than in overweight subjects (0.4 ± 0.2, P <.01 v normal). The significant fasting-induced GH increase in normal-weight men (to 5.6 ± 2.2 mU/L, P <.05 v basal) was inversely related to BMI ( r = −.86, P = .0006). GH pulse amplitudes but not frequencies were different for both groups and were increased by fasting in normal subjects but not in obese subjects. Plasma glucose showed comparable mean levels basally, and fasting induced a more pronounced decrease of mean glucose in normal men (−1.9 ± 0.6 v −1.4 ± 0.3 mmol/L in obese) with a negative correlation between changes in mean GH and glucose ( r = −.70, P = .022). IGF-I concentrations were similar in both groups and were unchanged with energy restriction, whereas IGF-II showed higher basal and fasting-associated values in obese subjects. The IGF-BC increased significantly during fasting in normal-weight men. In conclusion, nutritional regulation of GH release is associated with a modulation of GH pulse amplitudes, whereas GH pulse frequencies remain unchanged. These results suggest a stable pulse generation with amplitude modifications by direct substrate actions and/or metabolic influences of the somatostatinergic tone. Variations of free IGF-I and IGF-II are involved in the differential GH regulation of lean and obese subjects in periods of regular food intake. During short-term fasting, somatotrope GH release may also be influenced by changes in plasma glucose levels.

Bulk isolation of renal PCT and PST. I. Glucose-dependent metabolic differences

A new procedure for separately isolating milligram quantities of rabbit renal proximal straight (PST) or convoluted (PCT) tubules is described, and the differential abilities of these segments to utilize glucose as a metabolic substrate are investigated. Separate dissection of the cortical cortices and the outer medullary stripe, followed by collagenase digestion and discontinuous Percoll centrifugation, provide enriched populations (greater than 98% pure) of PCT (37 mg) and PST (14 mg), respectively, per rabbit. The purity of PCT and PST fractions was quantitated morphologically and by comparing the enriched activity of the proximal tubular marker leucine aminopeptidase and deenriched activity of the distal marker hexokinase to previously published values reported from microdissection studies. To investigate glucose-dependent metabolic differences, PCT and PST suspensions (1 mg/ml) were preincubated in Dulbecco's modified Eagle's-Ham's F-12 medium for 1 h before being incubated for 30 min in buffer with or without glucose as the only available metabolic substrate. In glucose-containing buffer, PST segments maintained their oxygen consumption and ATP contents at levels significantly higher than PCT segments. These differential responses between PST and PCT were glucose-dependent because they were abolished when segments were incubated under glucose-free conditions. Because responses in PCT were glucose-independent, these results suggest that PCT cannot utilize glucose to support oxidative metabolism, whereas PST segments can oxidatively metabolize this substrate. These differences in glucose utilization do not correlate with the distribution of glycolytic enzyme activities, suggesting that differential metabolic regulation of these enzymes may determine the ability of each segment to utilize glucose.