Citations

Insulin resistance increase with age but it is uncertain whether it is related to age or other factors. We measured insulin sensitivity by hyperinsulinemic euglycemic clamp in 48 men and women −12 each of young lean (YL) and obese (YO) as well as old lean (OL) and obese (OO) groups. OL (69.9±1.4 years), OO (71.0±1.5 years), YL (22.8±1.0 years) and YO (24.2±0.6 years) were matched for BMI. Older groups had lower peak VO 2 (32.4±1.3 ml/kg FFM) than young (46.9±1.0)(p<0.01) but no differences noted among the two older and younger groups. Similar glucose levels (90.4 to 91.4 mg/dL) were maintained during the glucose clamp. The glucose infusion rate (GIR) at steady state, was similar in the YO (38.7±5.4 mmol/kgFFM/min) and OO (43.0±2.4) where as YL (55.5±3.7) and OL (54.2±3.4) were higher (p<0.02) than obese groups. GIR was significantly correlated to fasting insulin levels (R 2 =0.32, p<0.001). Energy expenditure per FFM, was not different amongst the 4 groups. Adiponectin levels were higher in the older groups (12526±1109 ng/mL) than in the younger (8640±392, p<0.01). A trend toward lower adiponectin levels was seen in OO (10394 ± 1187 ng/ml) than OL (14657 ±1705, p=0.05). A similar trend was not seen comparing the YO to YL. However, the OL had a significantly higher adiponectin level than the YL (9023.8 ± 595 ng/mL, p<0.01). GIR was not significantly correlated to age (R 2 =0.00005, p=NS) nor to peak VO2 (R 2 =0.007, p=NS) but was significantly correlated to truncal fat (R 2 =0.17, p<0.01) and BMI (R 2 =0.25, p<0.001). The age‐related decline in insulin sensitivity is not accounted for by fitness levels or age per se. The results demonstrated that insulin resistance in aging is explained by change in body composition rather than age.

Insulin resistance is associated with skeletal muscle mitochondrial (mito) dysfunction in many conditions including type 2 diabetes (T2D). Poor glycemic control in T2D is associated with muscle mito gene transcript alterations. It is unknown whether muscle mito changes are related to reduced insulin action or vice versa. We obtained vastus lateralis muscle biopsies from 7 type 1 diabetic patients (T1D) following 8–12 hrs of insulin deprivation (I−) and replacement (I+) and measured skeletal muscle mito ATP production (MATP) and gene transcripts. I− and I+ changed glucose and glucagon levels (glucose=291±15mg/dl in I− vs 97±3 in I+ and glucagon=79.8±15.3 pg/mL in I− vs 47.3±5.9 in I+, p<0.04). Whole body O 2 consumption increased during I− by 15% (p<0.05) but maximal MATP decreased by 21.3–27.9% (p<0.05). Gene transcript profiling in skeletal muscle analyzed using Affymetrix GeneChip arrays showed alterations in 2355 transcripts. Pathway analysis showed down‐regulation of oxidative phosphorylation and arachidonic acid metabolism, and up‐regulation of VEGF and actin cytoskeleton signaling in I−. We conclude that the reduced insulin action that occurred during I− in T1D resulted in substantial changes in gene transcript levels of muscle mito genes and reduced maximal MATP, supporting that insulin is key regulator of muscle mito function by exerting its effect at various levels. Supported by NIHRO1DK41973.

Insulin regulates skeletal muscle protein degradation, but the types of proteins being degraded in vivo remain to be determined due to methodological limitations. We present a method to assess the types of skeletal muscle proteins that are degraded by extracting their degradation products as low-molecular weight (LMW) peptides from muscle samples. High-resolution mass spectrometry was used to identify the original intact proteins that generated the LMW peptides, which we validated in rodents and then applied to humans. We deprived insulin from insulin-treated streptozotocin (STZ) diabetic mice for 6 and 96 h and for 8 h in type 1 diabetic humans (T1D) for comparison with insulin-treated conditions. Protein degradation was measured using activation of autophagy and proteasome pathways, stable isotope tracers, and LMW approaches. In mice, insulin deprivation activated proteasome pathways and autophagy in muscle homogenates and isolated mitochondria. Reproducibility analysis of LMW extracts revealed that ∼80% of proteins were detected consistently. As expected, insulin deprivation increased whole body protein turnover in T1D. Individual protein degradation increased with insulin deprivation, including those involved in mitochondrial function, proteome homeostasis, nDNA support, and contractile/cytoskeleton. Individual mitochondrial proteins that generated more LMW fragment with insulin deprivation included ATP synthase subunit-γ (+0.5-fold, P = 0.007) and cytochrome c oxidase subunit 6 (+0.305-fold, P = 0.03). In conclusion, identifying LMW peptide fragments offers an approach to determine the degradation of individual proteins. Insulin deprivation increases degradation of select proteins and provides insight into the regulatory role of insulin in maintaining proteome homeostasis, especially of mitochondria.

OBJECTIVE Reductions in insulin sensitivity in conjunction with muscle mitochondrial dysfunction have been reported to occur in many conditions including aging. The objective was to determine whether insulin resistance and mitochondrial dysfunction are directly related to chronological age or are related to age-related changes in body composition. RESEARCH DESIGN AND METHODS Twelve young lean, 12 young obese, 12 elderly lean, and 12 elderly obese sedentary adults were studied. Insulin sensitivity was measured by a hyperinsulinemic-euglycemic clamp, and skeletal muscle mitochondrial ATP production rates (MAPRs) were measured in freshly isolated mitochondria obtained from vastus lateralis biopsy samples using the luciferase reaction. RESULTS Obese participants, independent of age, had reduced insulin sensitivity based on lower rates of glucose infusion during a hyperinsulinemic-euglycemic clamp. In contrast, age had no independent effect on insulin sensitivity. However, the elderly participants had lower muscle MAPRs than the young participants, independent of obesity. Elderly participants also had higher levels inflammatory cytokines and total adiponectin. In addition, higher muscle MAPRs were also noted in men than in women, whereas glucose infusion rates were higher in women. CONCLUSIONS The results demonstrate that age-related reductions in insulin sensitivity are likely due to an age-related increase in adiposity rather than a consequence of advanced chronological age. The results also indicate that an age-related decrease in muscle mitochondrial function is neither related to adiposity nor insulin sensitivity. Of interest, a higher mitochondrial ATP production capacity was noted in the men, whereas the women were more insulin sensitive, demonstrating further dissociation between insulin sensitivity and muscle mitochondrial function.

It remains to be determined whether systemic insulin replacement normalizes synthesis rates of different plasma proteins and whether there are differential effects on various plasma proteins. We tested a hypothesis that insulin deprivation differentially affects individual plasma protein synthesis and that systemic insulin treatment may not normalize synthesis of all plasma proteins. We measured synthesis rates of 41 plasma proteins in seven each of type 1 diabetic (T1DM) and nondiabetic participants (ND) using [ ring-13C6]phenylalanine as a tracer. T1DM were studied while on chronic insulin treatment and during 8 h insulin deprivation. Insulin treatment normalized glucose levels, but plasma insulin levels were higher during insulin treatment than during insulin deprivation in T1DM and ND. Individual plasma proteins were purified by affinity chromatography and two-dimensional gel electrophoresis. Only 41 protein gel spots from over 300 were chosen based on their protein homogeneity. Insulin deprivation and hyperglycemia either significantly increased ( n = 12) or decreased ( n = 12) synthesis rates of 24 of 41 plasma proteins in T1DM compared with ND. Insulin treatment normalized synthesis rates of 13 of these 24 proteins, which were altered during insulin deprivation. However, insulin treatment significantly altered the synthesis of 14 additional proteins. In conclusion, acute insulin deprivation caused both a decrease and increase in synthesis rates of many plasma proteins with various functions. Moreover, chronic systemic insulin treatment not only did not normalize synthesis of all plasma proteins but also altered synthesis of several additional proteins that were unaltered during insulin deprivation.

OBJECTIVE—Muscle mitochondrial dysfunction occurs in many insulin-resistant states, such as type 2 diabetes, prompting a hypothesis that mitochondrial dysfunction may cause insulin resistance. We determined the impact of insulin deficiency on muscle mitochondrial ATP production by temporarily depriving type 1 diabetic patients of insulin treatment. RESEARCH DESIGN AND METHODS—We withdrew insulin for 8.6 ± 0.6 h in nine C-peptide–negative type 1 diabetic subjects and measured muscle mitochondrial ATP production and gene transcript levels (gene array and real-time quantitative PCR) and compared with insulin-treated state. We also measured oxygen consumption (indirect calorimetry); plasma levels of glucagon, bicarbonate, and other substrates; and urinary nitrogen. RESULTS—Withdrawal of insulin resulted in increased plasma glucose, branched chain amino acids, nonesterified fatty acids, β-hydroxybutyrate, and urinary nitrogen but no change in bicarbonate. Insulin deprivation decreased muscle mitochondrial ATP production rate (MAPR) despite an increase in whole-body oxygen consumption and altered expression of many muscle mitochondrial gene transcripts. Transcript levels of genes involved in oxidative phosphorylation were decreased, whereas those involved in vascular endothelial growth factor (VEGF) signaling, inflammation, cytoskeleton signaling, and integrin signaling pathways were increased. CONCLUSIONS—Insulin deficiency and associated metabolic changes reduce muscle MAPR and expression of oxidative phosphorylation genes in type 1 diabetes despite an increase in whole-body oxygen consumption. Increase in transcript levels of genes involved in VEGF, inflammation, cytoskeleton, and integrin signaling pathways suggest that vascular factors and cell proliferation that may interact with mitochondrial changes occurred.

Abstract A case of granulomatous slack skin disease is presented in which we studied the possible involvement of extrarenal 1,25(OH)2D in the pathogenesis of the patient's hypercalcemia. Immunolocalization of 1α-OH in skin showed simultaneous dysregulation in epithelial and granulomatous cells. Introduction: Granuloma-forming diseases such as sarcoidosis are associated with extrarenal synthesis of active 1,25-dihydroxyvitamin D [1,25(OH)2D]. Here we describe a case of granulomatous slack skin disease in which we have studied the possible involvement of extrarenal synthesis of 1,25(OH)2D in the pathogenesis of the patient's hypercalcemia. The aim of the study was to clarify the etiology of hypercalcemia in this patient. Materials and Methods: This was a case study of a 19-year-old man with a T-cell lymphoproliferative disorder diagnosed as granulomatous slack skin disease who presented with hypercalcemia and raised serum 1,25(OH)2D. Analysis of expression of the enzyme 25-hydroxyvitamin D 1α-hydroxylase (1α-hydroxylase), which catalyzes synthesis of 1,25(OH)2D, was carried out by immunohistochemical analysis of involved and uninvolved skin. Approval was granted by the Mayo Foundation Institutional Review Board and Biospecimens Subcommittee. Results: In uninvolved skin, expression of 1α-hydroxylase was confined to the basal layer of the epidermis, whereas slack skin showed overexpression of the enzyme in dermal granulomata and basal cells of the epidermis. Conclusions: Hypercalcemia associated with granulomatous slack skin syndrome seems to be caused by dysregulation of 1α-hydroxylase expression in both epidermal and dermal granulomatous cells. This contrasts with psoriasis and sarcoidosis of the skin, in which overexpression of the enzyme is restricted to keratinocytes and granulomata, respectively.