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. 2009 Feb 26;23(6):881–892. doi: 10.1210/me.2008-0274

Increased Insulin Sensitivity in Mice Lacking Collectrin, a Downstream Target of HNF-1α

Sandra M Malakauskas 1, Wissam M Kourany 1, Xiao Yin Zhang 1, Danhong Lu 1, Robert D Stevens 1, Timothy R Koves 1, Hans E Hohmeier 1, Deborah M Muoio 1, Christopher B Newgard 1, Thu H Le 1
PMCID: PMC2691681  PMID: 19246514

Abstract

Collectrin is a downstream target of the transcription factor hepatocyte nuclear factor-1α (HNF-1α), which is mutated in maturity-onset diabetes of the young subtype 3 (MODY3). Evidence from transgenic mouse models with collectrin overexpression in pancreatic islets suggests divergent roles for collectrin in influencing β-cell mass and insulin exocytosis. To clarify the function of collectrin in the pancreas, we used a mouse line with targeted deletion of the gene. We examined pancreas morphology, glucose homeostasis by ip glucose tolerance testing (IPGTT) and insulin tolerance testing (IPITT), and pancreas function by in vivo acute-phase insulin response determination and glucose-stimulated insulin secretion from isolated islets. We find no difference in either pancreas morphology or function between wild-type and collectrin-deficient animals (Tmem27−/y). However, we note that by 6 months of age, Tmem27−/y mice exhibit increased insulin sensitivity by IPITT and decreased adiposity by dual-energy x-ray absorptiometry scanning compared with wild-type. We have previously reported that Tmem27−/y mice exhibit profound aminoaciduria due to failed renal recovery. We now demonstrate that Tmem27−/y animals also display inappropriate excretion of some short-chain acylcarnitines derived from amino acid and fatty acid oxidation. We provide further evidence for compensatory up-regulation of oxidative metabolism in Tmem27−/y mice, along with enhanced protein turnover associated with preserved lean mass even out to 1.5 yr of age. Our studies suggest that collectrin-deficient mice activate a number of adaptive mechanisms to defend energy homeostasis in the setting of ongoing nutrient losses.

Targeted deletion of collectrin in mice results in enhanced insulin sensitivity with age that is associated with upregulation of oxidative metabolism and decreased adiposity.

Type 2 diabetes mellitus is largely a polygenic disorder compounded by significant environmental effects. However, monogenic forms of the disease also exist. The most widely studied of these is maturity-onset diabetes of the young (MODY), which displays an autosomal dominant inheritance pattern and typically presents as clinically significant disease by the second decade of life (1). There are six subtypes of MODY, and most result from mutations in genes that encode pancreatic β-cell transcription factors (1). The most prevalent subtype is MODY3, which is associated with varied mutations in hepatocyte nuclear factor-1α (HNF-1α) (2), a transcription factor expressed in numerous tissues including pancreatic islets (3).

To explore the underlying disease mechanisms, a number of mouse models have been employed. Two models with targeted deletion of HNF-1α reveal a number of abnormal features related to pancreatic physiology: hyperglycemia with reduced pancreatic insulin content (4,5), decreased insulin levels in response to both glucose and arginine stimulation (4), and decreased β-cell ATP production arising from impaired glycolytic signaling (6). Other transgenic mouse (7) and β-cell model systems (8) have been generated that express the HNF1α-P291fsinsC mutation, the most common mutation associated with MODY3 (9). Consistent with the phenotype of the HNF-1α knockout mouse lines, these models demonstrate defects in glucose-stimulated insulin secretion (7,8), pancreatic insulin content (7), β-cell proliferation (7), and mitochondrial ATP production (8).

Despite the insights gained from these model systems, many mechanistic details regarding the role of HNF-1α in the pathogenesis of MODY3 remain unknown, and efforts have focused on identifying causative downstream targets of HNF-1α. Recently, two groups have independently identified the gene collectrin as a novel target (10,11). Collectrin was originally described in a rodent model of chronic kidney disease (12,13). It is a transmembrane protein with close homology to ACE2 (13). Collectrin is highly conserved among species (13) and has wide tissue distribution (www.genecards.org/cgi-bin/carddisp.pl?gene= TMEM27). To elucidate its function in the pancreas, Akpinar et al. (10) used in vitro cell model systems and a transgenic mouse line overexpressing collectrin under the control of the rat insulin promoter. Transgenic mice exhibited increased β-cell mass and insulin content with normal glucose tolerance tests. Using similar techniques and an independent transgenic model, Fukui et al. (11) provided evidence that collectrin mediates insulin exocytosis via interaction with the SNARE complex. In contrast to the findings of Akpinar et al., the transgenic mice of Fukui et al. exhibited no difference in β-cell mass but had enhanced glucose tolerance. Given the disparate findings of these two studies, we sought to clarify the role of collectrin in the pancreas by using a mouse model with targeted deletion of collectrin (14). Here, we demonstrate that collectrin-deficient mice display no difference in pancreatic phenotype compared with wild-type animals but exhibit increased peripheral insulin sensitivity and decreased adiposity associated with urinary losses of intermediate metabolites of amino acid and fatty acid oxidation.

Results

Generation of collectrin-deficient mouse line and baseline phenotypic characteristics

We generated a mouse line on a mixed genetic background (C57BL/6 and 129/SvEv) with global disruption of the collectrin gene (Tmem27, NX-17), as previously described (14). The experiments described herein were conducted at ages 2–8 months. All animals were fed on standard rodent laboratory chow before the studies.

Both wild-type (Tmem27+/y) and collectrin-deficient (Tmem27−/y) mice display similar baseline body weights at 2 months of age (Fig. 1A). However, by 6 months, Tmem27−/y animals have significantly decreased body weight compared with control (Fig. 1A; Tmem27+/y 36.8 ± 1.6 g vs. Tmem27−/y 32.3 ± 0.9 g; P = 0.02). This is despite the trend toward increased food intake in the collectrin-deficient group (Tmem27+/y 1.1 ± 0.1 g vs. Tmem27−/y 1.3 ± 0.1 g; P = 0.069). We have noted this trend in our previous studies in which we characterized the renal phenotype of the collectrin-deficient mice (14). In the prior study, we also demonstrated that loss of collectrin results in primary renal wasting of amino acids due to a proximal tubule defect (14), and we have speculated that the mild hyperphagia is compensatory for nutrient losses. To further assess the differences in body weight in the older cohort of animals, we examined body composition by dual-energy x-ray absorptiometry (DEXA) and find that collectrin-deficient animals have approximately 25% less body fat than wild-type (Fig. 1B; Tmem27+/y 23.9 ± 1.8% vs. Tmem27−/y 18.1 ± 0.9%; P = 0.009).

Figure 1.

Figure 1

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Tmem27−/y mice display lower body weight and percent body fat at 6 months of age. A, Body weights at 2 and 6 months of age on control diet. n ≥ 5. *, P = 0.02 vs. Tmem27+/y at 6 months. B, Body fat composition measured by DEXA scanning at 6 months of age. n = 14. *, P = 0.009 vs. Tmem27+/y. Data represent the mean ± sem.

Glucose and insulin levels in the fasting state

We next assessed measures of glucose homeostasis in the fasting state, examining cohorts of animals in both younger and older age groups. There was no difference in fasting blood glucose between Tmem27+/y and Tmem27−/y animals in either age group (Fig. 2A). Fasting serum insulin levels were assessed under similar conditions. We observed increases in serum insulin levels at 6 months compared with 2 months within each genotypic cohort (Fig. 2A), as we would expect with development of age-related insulin resistance (15). However, although no difference was noted between genotypic groups at 2 months of age (Fig. 2A), we observed reduced fasting serum insulin levels in collectrin-deficient mice compared with wild-type by 6 months (Fig. 2A; Tmem27+/y 1.16 ± 0.18 ng/ml vs. Tmem27−/y 0.75 ± 0.08 ng/ml; P = 0.05).

Figure 2.

Figure 2

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Tmem27−/y mice exhibit evidence of enhanced insulin sensitivity by in vivo assessments of glucose homeostasis at 6 months. A, Blood glucose and serum insulin values from animals fasted overnight. Blood glucose was measured using a glucometer, and serum insulin was quantified by ELISA. n ≥ 5. *, P = 0.05, Tmem27+/y vs. Tmem27−/y at 6 months; +, P = 0.02, Tmem27+/y at 2 vs. 6 months; **, P = 0.04, Tmem27−/y at 2 vs. 6 months. B, IPGTT at 2 months of age. Animals were fasted for 6 h and assessed for response to 1.5 g/kg glucose: left, blood glucose values; right, corresponding serum insulin levels. n ≥ 7. C, IPGTT repeated at 6 months of age: left, blood glucose values; right, corresponding serum insulin levels. n = 19. *, P ≤ 0.04 vs. Tmem27+/y at the corresponding time point. D, IPITT at 2 months of age (left, n = 9) or 6 months of age (right, n = 10). Animals were fasted for 6 h, and the blood glucose levels were monitored in response to 0.75 U/kg insulin. *, P ≤ 0.05 vs. Tmem27+/y at the corresponding time point. Data represent the mean ± sem.

In vivo glucose and insulin tolerance testing

To evaluate the mechanisms underlying the differences in measured serum insulin levels, we assessed glucose homeostasis in wild-type and collectrin-deficient animals at each age. Intraperitoneal glucose tolerance testing (IPGTT) was performed after maintenance on normal chow. After 1.5 g/kg glucose bolus, blood glucose and serum insulin measurements were made at time points from 0–60 min. At 2 months of age, no differences were noted in glucose or insulin levels during the IPGTT (Fig. 2B). Area under the curve (AUC) for glucose (Tmem27+/y 12,012 ± 709 mg/min · dl vs. Tmem27−/y 12,055 mg/min · dl ± 823; P value not significant) and insulin (Tmem27+/y 30.6 ± 3.4 mg/min · ml vs. Tmem27−/y 30.3 ± 5.2 mg/min · ml; P value not significant) were equivalent between groups. However, after 6 months, differences emerged. Although collectrin-deficient animals exhibited similar glucose clearance compared with wild-type (Fig. 2C), they displayed lower insulin levels compared with control (Fig. 2C; Tmem27+/y AUC-insulin 91.8 ± 18.4 mg/min · ml vs. Tmem27−/y AUC-insulin 51.8 ± 6.7 mg/min · ml; P = 0.05). This suggests increased insulin sensitivity.

To more definitively assess response to insulin, wild-type and collectrin-deficient animals were subjected to ip insulin tolerance testing (IPITT) with 0.75 U/kg insulin administration (Fig. 2D). Consistent with the findings of the IPGTT, there was no difference in response to insulin between the two groups at 2 months of age. However, by 6 months of age, the collectrin-deficient mice exhibited a greater decrease from baseline in serum glucose levels compared with wild-type at 15 min (P = 0.01), 30 min (P = 0.05), 60 min (P = 0.04), and 90 min (P = 0.05) after ip insulin bolus. These data are consistent with greater peripheral insulin sensitivity in the older cohort of Tmem27−/y mice compared with age-matched controls.

Measures of insulin resistance

To further assess differences in peripheral insulin sensitivity, we next determined the insulin resistance index (IR) (16) using the areas under the glucose and insulin curves obtained from the 1.5 g/kg IPGTT (IR = AUCglucose × AUCinsulin). Collectrin-deficient animals displayed a statistical trend toward a lower IR value compared with wild-type (Fig. 3A; Tmem27+/y 11.7 × 105 ± 2.4 × 105 vs. Tmem27−/y 6.5 × 105 ± 1.0 × 105; P = 0.06). We also calculated the homeostasis model assessment of insulin resistance (HOMA-IR) (17) using fasting blood glucose and serum insulin levels [HOMA-IR = fasting glucose (millimoles per liter) × fasting insulin (microunits per milliliter)/22.5]. Here, collectrin-deficient mice exhibited a statistically significant decrease over wild-type (Fig. 3B; Tmem27+/y 13.5 ± 2.7 vs. Tmem27−/y 5.4 ± 1.0; P = 0.02). This is consistent with our IPITT data.

Figure 3.

Figure 3

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Calculated indices of insulin resistance at 6 months are lower in Tmem27−/y mice. A, The insulin resistance index was calculated from the areas under the blood glucose and serum insulin curves obtained during the 1.5 g/kg IPGTT at 6 months (IR = AUCglucose × AUCinsulin). n = 19. †, P = 0.06 vs. Tmem27+/y. B, HOMA-IR was determined from fasting blood glucose and serum insulin levels at 6 months [HOMA-IR = fasting glucose (millimoles per liter) × fasting insulin (microunits per milliliter)/22.5]. n = 9. *, P = 0.02 vs. Tmem27+/y. Data represent the mean ± sem. Both indices are reported in arbitrary units.

Assessment of pancreatic morphology

We next evaluated the possibility of an underlying alteration in pancreatic physiology contributing to our phenotype. Earlier studies have suggested that collectrin overexpression results in a significant pancreatic phenotype, with possible enhancement in β-cell mass (10) and insulin exocytosis (11). We, therefore, assessed measures of pancreatic morphology and function in the wild-type and collectrin-deficient cohorts. We evaluated pancreata isolated from animals maintained on normal chow (NC) or high-fat diet (HFD) for 18 wk. Our inclusion of HFD derives from prior evidence of enhanced collectrin expression in islets isolated from rodent models of diet-induced obesity (11). Gross morphological examination of hematoxylin-stained pancreatic sections revealed no obvious differences between genotypic groups under either dietary condition (Fig. 4). Quantitative morphometric analysis of these sections further confirmed no statistically significant difference between the cohorts with respect to pancreas-to-body weight ratio, islet number, relative β-cell area, or β-cell proliferation (Table 1).

Figure 4.

Figure 4

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There are no gross morphological differences between the pancreata from Tmem27+/y and Tmem27−/y mice on NC and HFD. Hematoxylin staining of pancreatic sections at ×20 is shown. Islets are identified by insulin immunostaining in red. Top, NC: left, pancreas from Tmem27+/y animal; right, pancreas from Tmem27−/y animal. Bottom, HFD: left, pancreas from Tmem27+/y animal; right, pancreas from Tmem27−/y animal.

Table 1.

Morphometric analysis and β-cell proliferation assessment of the pancreas under normal and high-fat dietary conditions

 NC
HFD
Tmem27+/y Tmem27−/y Tmem27+/y Tmem27−/y
Pancreas/body weight (mg/g) 5.58 ± 0.26 5.81 ± 0.14 8.17 ± 0.41 8.01 ± 0.53
Islet number/pancreas area (mm−2) 0.22 ± 0.02 0.18 ± 0.02 0.98 ± 0.31 1.63 ± 0.73
β-Cell/pancreas area (%) 0.35 ± 0.04 0.34 ± 0.07 1.16 ± 0.38 1.41 ± 0.43
BrdU-positive cells (%) 0.72 ± 0.08 0.54 ± 0.07 3.1 ± 0.9 3.5 ± 1.3
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There are no observed differences between genotypic cohorts at 6 months of age when compared within dietary groups. For morphometric analyses, measures were averaged over three stepped sections per mouse. For BrdU assay, at least 5000 insulin-positive cells were surveyed. Reported values are means ± sem. n ≥ 4. 

In vivo acute-phase insulin response at 6 months

To evaluate pancreatic function in vivo, we assessed the response of serum insulin levels to stimulation with ip injection of 3 g/kg glucose or 0.3 g/kg arginine, monitoring at 0, 2, 5, 15, 30, and 60 min. Interestingly, glucose stimulation failed to elicit an acute-phase insulin response in either wild-type or collectrin-deficient mice (Fig. 5A). This is expected given the strains that comprise the genetic background of our mouse model (18,19). However, arginine stimulation effectively induced an acute-phase insulin response in both groups that appeared similar in magnitude (Fig. 5B). For more direct comparison and to correct for the decreased basal insulin levels in collectrin-deficient mice, we normalized the peak serum insulin levels at 2 min to the corresponding basal levels at 0 min and observed a similar fold increase between genotypic groups (Tmem27+/y 1.6 ± 0.3 vs. Tmem27−/y 1.8 ± 0.2; P = 0.57).

Figure 5.

Figure 5

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Tmem27−/y mice display a similar acute-phase insulin response to glucose and arginine stimulation as compared with Tmem27+/y mice at 6 months. A, Animals were fasted for 6 h, and serum insulin levels were monitored in response to an ip injection of 3 g/kg glucose. Although Tmem27−/y animals exhibit lower absolute serum insulin levels, the acute phase is suppressed in both groups. n = 8. *, P ≤ 0.05 vs. Tmem27+/y at corresponding time points. B, Animals were fasted for 6 h, and serum insulin levels were monitored in response to an ip injection of 0.3 g/kg arginine. Both groups exhibit a similar fold change in peak insulin response at 2 min compared with baseline. n = 6. Data represent the mean ± sem.

Insulin secretion from isolated islets

We next examined islet function using isolated islets from 6-month-old animals maintained on NC and HFD (Fig. 6). There were no differences in insulin secretion in response to 5.5 and 16.7 mm glucose stimulation in Tmem27+/y vs. Tmem27−/y animals on either diet (Fig. 6A). Similarly, no difference was noted when insulin response at 16.7 mm glucose was expressed relative to the response at 5.5 mm glucose (Fig. 6B).

Figure 6.

Figure 6

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There is no difference in isolated islet function on NC and HFD at 6 months of age. A, Islets were isolated from Tmem27+/y and Tmem27−/y animals by Liberase R1 enzyme digestion, maintained in culture, and stimulated with 5.5 and 16.7 mm glucose. Secreted insulin was determined by RIA and normalized to total cellular protein content. Left, Control diet; right, HFD. B, Relative increase in insulin secretion with 16.7 mm glucose compared with 5.5 mm glucose on control diet and HFD. Data represent the mean ± sem. of two independent experiments, each of which was performed in triplicates of 20 islets per condition collected from four animals of each genotype.

Assessment of amino acid metabolism

We and others have previously demonstrated that collectrin-deficient animals waste amino acids in their urine due to failed recovery at the proximal tubule (14,20). Given the importance of amino acids to energy homeostasis and their influence on insulin sensitivity and signaling (21), we hypothesize that loss of amino acids in collectrin-deficient animals contributes to the enhanced insulin sensitivity phenotype we observe. To examine this possibility further, we profiled fasting plasma acylcarnitines in 6 month-old wild-type and collectrin-deficient mice. Acylcarnitines are formed as byproducts of fatty acid and amino acid oxidation, enabling the transfer of acyl-coenzyme A (CoA) species with varying chain lengths into and out of the mitochondrion (22). Short-chain acylcarnitines with three, four, and five carbons derive from branched-chain amino acids (BCAA) as well as odd- and even-chain fatty acids (23). Acetylcarnitine (C2) is formed from acetyl-CoA, the end product of all oxidative substrate metabolism (24). Therefore, acylcarnitine profiling can provide substantial information regarding the activities of the various oxidative pathways. Although we observed no significant differences in medium- and long-chain acylcarnitines between genotypes, collectrin-deficient mice displayed marked decreases in free carnitine (FC) (54% reduction, P < 0.0001) and a number of short-chain species, specifically C2 (59% reduction, P < 0.0001), C3 (47% reduction, P < 0.0001), C4 (49% reduction, P = 0.0011), C4-OH (31% reduction, P = 0.079), and C4-DC (66% reduction, P = 0.019) (Fig. 7A).

Figure 7.

Figure 7

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Tmem27−/y animals exhibit decreased plasma levels of a number of short-chain acylcarnitine species with corresponding urinary losses of a subset of those species. A, Plasma FC and acylcarnitine levels from animals fasted overnight were measured by mass spectrometry. n = 13–14. *, P < 0.0001 (FC, C2, C3), P = 0.0011 (C4), and P = 0.019 (C4-DC) vs. Tmem27+/y. B, The 24-h urine excretion of FC and acylcarnitines normalized to creatinine. Animals were housed individually with free access to food and water in metabolic cages for 24-h urine collection. FC, acylcarnitines, and creatinine were quantified by mass spectrometry. n = 11–12. *, P ≤ 0.05 vs. Tmem27+/y. C, Fractional excretions (FE) of FC and acylcarnitines were calculated using acylcarnitine and creatinine concentrations obtained from spot plasma and urine samples collected after an overnight fast. FE (percent) = ([AC]urine × [Cr]plasma)/([AC]plasma × [Cr]urine). C3 and C5:1: n = 5–8. *, P ≤ 0.05 vs. Tmem27+/y. C4-DC: n = 3–5. †, P = 0.07 vs. Tmem27+/y. Data represent the mean ± sem.

It is known that the renal proximal tubule plays an important role in reabsorption of filtered FC and short-chain acylcarnitines (SC-AC) (25). Because of the profound aminoaciduria in collectrin-deficient mice (14), we posited that these animals display a more generalized defect in proximal tubule function and also waste FC and SC-AC. Mice were housed in metabolic cages with free access to food for 24-h urine collection. FC and SC-AC were quantified using mass spectrometry analysis and normalized for creatinine excretion (Fig. 7B). To exclude dietary effects, we collected plasma and spot urine samples under fasted conditions and quantified acylcarnitine (AC) and creatinine (Cr) for determination of the fractional excretions [FE = ([AC]urine × [Cr]serum)/([AC]serum × [Cr]urine)] (Fig. 7C). We note a statistically significant increase in excretion of C3 and C5:1 (84 and 62% increase, respectively, by fractional excretion, P ≤ 0.05) and a trend toward enhanced excretion of C4-DC (335% increase by fractional excretion, P = 0.072) in collectrin-deficient animals, supporting an inappropriate loss of these metabolites.

Alternative nutrient substrate utilization

The loss of oxidative intermediates we observe is not a generalized phenomenon, however, as recovery of FC and C2 is preserved. C2 is derived from acetyl-CoA, which in turn is generated from a number of metabolic processes, including amino acid and fatty acid oxidation as well as glycolysis via pyruvate. Although acetyl-CoA has numerous metabolic fates, it is a key substrate for the tricarboxylic acid (TCA) cycle and for ketogenesis. We hypothesize that Tmem27−/y mice up-regulate flux through energy-generating pathways to compensate for the loss of energetically important metabolites into the urine and that C2 levels in plasma fall as more acetyl-CoA is shunted to downstream pathways. To evaluate this possibility, we calculated the C2/SC-AC ratio and observe that it is significantly decreased in Tmem27−/y animals compared with wild-type (Fig. 8A; Tmem27+/y 16.5 ± 0.9 vs. Tmem27−/y 10.7 ± 1.3; P = 0.0013). We further observe that Tmem27−/y mice display increased serum ketones with fasting (Fig. 8B; Tmem27+/y 0.30 ± 0.04 mm vs. Tmem27−/y 0.50 ± 0.07 mm; P = 0.039).

Figure 8.

Figure 8

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Tmem27−/y animals display evidence for increased oxidative metabolism by a decreased plasma C2-AC/short-chain acylcarnitine ratio and increased fasting serum ketone level. A, Plasma C2 acylcarnitine concentration was normalized to the total short-chain species concentration as determined from fasting plasma samples. n = 13–14. *, P = 0.0013 vs. Tmem27+/y. B, Serum nonesterified free fatty acids and ketones were measured enzymatically with an autoanalyzer after an overnight fast. n = 6–9. *, P = 0.039 vs. Tmem27+/y. Data represent the mean ± sem.

Enhanced protein turnover

We next addressed the question of whether collectrin-deficient animals also employ compensatory responses specifically to counter amino acid wasting. We have previously reported that Tmem27−/y mice exhibit hyperphagia and maintain normal plasma amino acid levels in the fed state (14). Similarly, after an overnight fast, normal plasma amino acid levels are also observed (Fig. 9A). This would suggest enhanced muscle proteolysis to maintain plasma amino acid levels and provide substrate for amino acid catabolism. We therefore measured muscle amino acid content and found a statistical trend toward decreased levels (Fig. 9B). Because amino acid oxidation occurs primarily in the liver, we also measured amino acid content in the liver and confirmed the anticipated increase in Tmem27−/y animals (Fig. 9C).

Figure 9.

Figure 9

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Tmem27−/y animals maintain normal fasting plasma amino acid levels in the setting of decreased muscle and increased liver amino acid content. A, Plasma amino acid concentration was measured by mass spectrometry after overnight fast. n = 5–6. *, P ≤ 0.05 vs. Tmem27+/y. B, Muscle amino acid content in the fed state as determined by mass spectrometry, normalized for total protein, and expressed relative to wild-type. n = 5–6. †, P = 0.06 (Leu/Ile), and P = 0.07 (Glu) vs. Tmem27+/y. C, Liver amino acid content in the fed state as determined by mass spectrometry, normalized for total protein, and expressed relative to wild-type. n = 5–6. *, P = 0.02 (Leu/Ile) vs. Tmem27+/y; †, P = 0.08 (Met), and P = 0.06 (Phe) vs. Tmem27+/y. Data represent the mean ± sem.

To evaluate muscle protein turnover in more detail, we next measured 24-h urine 3-methylhistidine in the fed state. 3-Methylhistidine is a byproduct of myofibrillar protein breakdown and is freely and completely excreted in the urine (26). It is estimated that approximately 75% of the 3-methylhistidine pool derives from skeletal muscle (27). We note a significant increase in 3-methylhistidine excretion by collectrin-deficient mice (Fig. 10A; Tmem27+/y 277.2 ± 28.0 nmol/mg creatinine vs. Tmem27−/y 576.8 ± 29.0 nmol/mg creatinine; P < 0.0001). We reason that these animals would require chronic up-regulation of proteolysis in the setting of ongoing urinary amino acid losses, so we next evaluated Tmem27−/y mice for evidence of wasting of lean tissue mass. We performed DEXA scans on wild-type and collectrin-deficient mice at 1.5 yr of age and noted similar lean mass in both groups (Fig. 10B). Furthermore, we observed no differences in gastrocnemius-to-body weight ratios (Fig. 10C). These results imply a concomitant increase in protein synthesis.

Figure 10.

Figure 10

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Tmem27−/y animals display enhanced proteolysis yet maintain their lean mass. A, The 24-h urinary 3-methylhistidine excretion normalized to creatinine. Animals were housed individually with free access to food and water in metabolic cages for 24-h urine collection. 3-Methylhistidine was determined by autoanalyzer. n = 6. *, P < 0.0001 vs. Tmem27+/y. B, Lean body mass in 1.5-yr-old mice, measured by DEXA scanning. n = 8. C, Gastrocnemius-to-body weight ratios assessed in 1.5-yr-old animals. n = 8. Data represent the mean ± sem.

Discussion

We have demonstrated that mice lacking the collectrin gene exhibit increased insulin sensitivity that is associated with up-regulation of oxidative metabolism. Previously, two groups studying molecular mechanisms of MODY3 have proposed important functions for collectrin in pancreas physiology (10,11). Using transgenic mouse models with overexpression of collectrin in pancreatic islets, they have provided evidence to suggest that collectrin influences β-cell mass (10) and insulin exocytosis (11). Given these divergent findings, we attempted to clarify collectrin’s function by evaluating pancreatic structure and function in the collectrin-deficient mouse line. To assess structure, we performed morphometric analysis on pancreata isolated from wild-type and collectrin-deficient mice, comparing the effects of NC and HFD (Table 1). Collectrin transcript has been reported to be increased in islets from animals with diet-induced obesity (11). Furthermore, our mouse line is comprised, in part, of the C57BL/6 genetic background, and this strain is commonly used in models of diet-induced obesity. Although we observed the expected increase in morphometric measures with HFD compared with NC, we did not detect any differences between genotypes.

We next evaluated pancreatic function both in vivo and in vitro. We performed assessments of the acute-phase insulin response with administration of 3 g/kg glucose or 0.3 g/kg arginine (Fig. 5). Interestingly, we observed that both wild-type and collectrin-deficient mice exhibited a depressed acute-phase response when stimulated with glucose. This finding is not unexpected in the context of the genetic background, which is a mix of C57BL/6 and 129/SvEv. It is well documented that 129 substrains display blunted or absent acute-phase insulin response to glucose stimulation (18,19), and it is likely that influences from the 129 background predominate in our system. The 129 substrains do appropriately respond to ip arginine stimulation (19), and we observe this response in our mouse line. Insulin release must be considered in the context of insulin sensitivity, and so we corrected the peak insulin response at 2 min to the basal levels and found no difference between genotypic groups. We also examined islet function in vitro by glucose-stimulated insulin release assays (Fig. 6). Here, genetic background effects would have less influence. We chose a high-dose glucose stimulation and incubation time that would result in release of both first- and second-phase insulin. Under these conditions, islets from 129 substrains behave similarly to those from C57BL/6 mice (18). Again, we noted no differences between genotypes under either NC or HFD dietary conditions.

Overall, we detected no differences in measures of pancreatic morphology, β-cell proliferation, or insulin secretion between wild-type and collectrin-deficient animals, demonstrating that loss of collectrin function in vivo does not result in any detectable pancreatic phenotype. However, our data do not exclude the possibility that collectrin has a functional role in the pancreas, because compensatory mechanisms could have masked the effects of collectrin deletion. We have mentioned that collectrin has homology to ACE2 (13) and have performed preliminary quantitative PCR experiments to evaluate relative differences in ACE2 transcript levels between total pancreatic mRNA pools isolated from wild-type and collectrin-deficient mice. We do not appreciate a difference, but it is possible that a difference might be detectable in islet-rich preparations. Thus, those compensatory pathways that may be exerting influences on pancreatic function in our mouse line are not yet identified.

Despite the apparent absence of a pancreatic phenotype in the collectrin-deficient mice, we provide evidence for altered glucose homeostasis. By 6 months of age, the collectrin-deficient mice display increased peripheral insulin sensitivity as compared with wild-type. First, we observe that collectrin-deficient mice have lower fasting serum insulin levels than wild-type while maintaining similar blood glucose levels (Fig. 2A). This would suggest an enhancement in insulin sensitivity rather than a defect in insulin secretion. With the latter, we would expect increased blood glucose levels. Additionally, we have evaluated pancreatic function and detected no differences. Interestingly, both genotypic groups exhibit an increase in their fasting serum insulin levels at 6 months compared with 2 months. This observation is anticipated in the context of aging-associated insulin resistance (15). Therefore, one interpretation for the decreased fasting serum insulin levels in collectrin-deficient mice is that these animals are protected against the development of insulin resistance. Consistent with this is our observation that collectrin-deficient mice are also resistant to age-induced obesity (Fig. 1). In further support of altered insulin sensitivity, we performed IPGTT and IPITT experiments (Fig. 2, B–D). AUCinsulin obtained from the IPGTT data is significantly lower in collectrin-deficient mice, whereas AUCglucose is unchanged compared with wild-type. Again, both genotypic groups display increased insulin levels with aging, but this is less pronounced in collectrin-deficient animals. Additionally, wild-type animals are less responsive to exogenous insulin during ITT at 6 months, whereas collectrin-deficient animals respond similarly at 6 months as at 2 months. Therefore, collectrin-deficient mice maintain their insulin sensitivity, such that it is enhanced over wild-type by 6 months. Consistent with these findings, we also determined that collectrin-deficient mice exhibit lower indices of insulin resistance than wild-type (Fig. 3).

We have previously demonstrated that loss of collectrin in the kidney results in aminoaciduria secondary to impaired recovery at the proximal tubule, associated with altered membrane populations of amino acid transporters (14). It is well established that amino acids are critical to the regulation of energy homeostasis in mammals, in which they serve as both signaling molecules and substrates for diverse metabolic pathways (21). We therefore sought to examine the possibility of altered amino acid metabolism in collectrin-deficient animals. As a marker of amino acid metabolism, we measured plasma acylcarnitines and found significant decreases in FC and a number of short-chain species that can derive from BCAA catabolism (Fig. 7). There are a number of possible explanations for this observation. First, there could be impairment in amino acid oxidation itself. This is doubtful given the fact that we do not observe increases in any acylcarnitine species, as would be expected proximal to a metabolic blockade.

Second, decreased FC and SC-AC species could arise from loss into the urine. It is known that the renal proximal tubule plays a critical role in recovery of these energetically important species from the glomerular filtrate (25). Indeed, we demonstrated enhanced fractional excretion of some SC-AC species in the urine of collectrin-deficient animals (Fig. 7C), which would be consistent with a wasting phenomenon. There are reports that the kidney tubular epithelium can secrete acylcarnitines into the urine (28), and we cannot completely exclude this possibility in the collectrin-deficient animals. However, we would expect enhanced secretion under conditions in which there is increased renal generation of acylcarnitines. The literature supports such a scenario under conditions of renal injury with mitochondrial dysfunction, such as ischemia (29). We have no data to suggest such a mechanism in our mouse line and have previously reported normal renal histology and preserved creatinine clearance in Tmem27−/y mice (14). Additionally, wasting of SC-AC would be consistent with our prior report of failed amino acid recovery and would parallel human disease correlates. As mentioned previously, collectrin is a downstream target of transcription factor HNF-1α, loss of which in the kidney results in a renal Fanconi syndrome with generalized impairment of proximal tubule reabsorptive capacity (30). Similar to our mouse line, HNF-1α−/− animals display urinary wasting of amino acids (30). Human patients with Fanconi syndrome are reported to waste not only amino acids but also acylcarnitines (31). Additionally, proximal tubule transporters (OCTN1-3) have been identified within the apical brush border that can facilitate transport of FC and SC-AC (32).

Although many SC-AC are elevated in the urine, it is noteworthy that the enhanced SC-AC excretion is not generalized (Fig. 7). Fractional excretion of FC, C2, and C4 are similar to wild-type, suggesting that collectrin-deficient animals recover these species appropriately. Therefore, a third possible explanation for the decreased plasma levels observed is increased downstream utilization. We have demonstrated that Tmem27−/y mice have a decreased plasma C2/SC ratio. Because the main fate of C2 is the TCA cycle, these data would suggest enhanced downstream utilization in the TCA cycle. This would be consistent with the enhanced insulin sensitivity phenotype we observe. Additionally, Tmem27−/y mice exhibit increased fasting ketogenesis, which also consumes acetyl-CoA and indicates increased fatty acid oxidation (FAO). In support of increased FAO, collectrin-deficient mice fail to gain as much adiposity with aging as do wild-type (Fig. 1). This suggests that collectrin-deficient animals are up-regulating both carbohydrate and lipid oxidation. Although it is possible that ketogenesis can result from impairment in the TCA cycle with subsequent shunting of acetyl-CoA, this is unlikely. We have measured urine pyruvate and citrate as markers of TCA function and observe no difference compared with wild-type. Also, ketogenesis can be increased in settings of tissue glycogen depletion. We have measured liver and muscle glycogen levels and do not detect any differences between genotypic groups. Finally, it is possible that the decreased SC-AC could be entirely explained by up-regulation of FAO. However, we have measured liver and muscle acylcarnitines in the fed state when FAO would be low and observe profiles that statistically trend to reflect what we have reported in the serum. This would imply an increase in TCA utilization. Therefore, we propose a model in which collectrin-deficient animals enhance flux through carbohydrate and lipid oxidative pathways to compensate for urinary losses of amino acids and short-chain oxidative metabolites.

We have proposed that increased oxidative flux may, at least in part, provide an explanation for the enhanced insulin sensitivity we observe. Whether or not enhanced oxidation is associated with insulin resistance or sensitivity depends on the degree to which nutrient supply and demand are matched. In obesity and insulin resistance, in which the oxidative machinery becomes overloaded by substrate or there is failure to up-regulate the TCA cycle, toxic lipid intermediates can impair insulin signaling (33,34). On the other hand, conditions of increased substrate demand, such as endurance exercise, result in increased insulin sensitivity and increased oxidative flux (35). We propose that collectrin-deficient mice exhibit increased substrate demand due to nutrient losses in the urine. However, it is not clear whether amino acid or SC-AC wasting has the predominant effect. It will be difficult to distinguish these processes, because amino acid oxidation in and of itself produces SC-AC. It is evident from the literature that impaired BCAA metabolism is sufficient to produce enhanced insulin sensitivity. The branched-chain aminotransferase (BCATm)-deficient mouse line (36) is unable to metabolize branched-chain amino acids and exhibits increased insulin sensitivity and decreased adiposity similar to collectrin-deficient mice. Interestingly, these animals also display a futile cycle of protein turnover (36) yet maintain their lean mass. Likewise, we have provided data to suggest a similar response in collectrin-deficient animals and, in further observations, find no evidence of muscle wasting in animals as a function of aging out to 1.5 yr.

The question remains whether or not loss of collectrin makes a direct contribution to our observed phenotype or induces indirect up-regulation of pathways that defend nutrient homeostasis. There is increasing evidence that collectrin in the kidney affects the surface membrane targeting and/or stabilization of various transporters, including those for amino acids (14,20) and sodium (37) and various cilia-associated proteins (38). Indeed, collectrin localization appears largely limited to the cell surface and subapical vesicles (10,11,14,20,38). However, the function of collectrin in other tissues is unknown and may be different from in the kidney. We have performed Western blotting analysis of membrane fractions from liver and muscle to examine whether the amino acid transporters reduced in the kidneys of collectrin-deficient mice are similarly affected in these other tissues. We did not detect a difference from wild-type. Therefore, we conclude that failed cellular uptake of amino acids likely manifests only in the kidney. It is possible that collectrin directs trafficking of other, as of yet unidentified, transporters in other tissues and that this contributes to our phenotype. Additionally, it is conceivable that collectrin could modulate surface transporter or receptor function. Collectrin is predicted to have a calmodulin binding site in its C terminus (39) and so may be involved in calcium signaling. Finally, because collectrin has homology to ACE2, it is possible that our phenotype is influenced by alterations in renin-angiotensin system (RAS) function. It is well documented that the RAS can modulate insulin sensitivity (40). We have, therefore, done a preliminary phenotypic evaluation to assess for altered RAS activity in our mouse model. We have measured sodium and potassium excretion and blood pressure under different dietary sodium conditions and do not detect differences compared with wild-type. Here again, genetic background effects could be masking a phenotype. In our experience, C57BL/6 mice are resistant to salt-induced hypertension.

Herein we have described the metabolic phenotype of collectrin-deficient mice. We propose a model in which loss of collectrin results in failed renal recovery of energetically critical metabolites, such as amino acids and short-chain acylcarnitines. The resultant adaptive responses in these animals lead to up-regulation of energy-generating processes, including carbohydrate and lipid oxidation, that are associated with enhanced insulin sensitivity and decreased adiposity. These animals also display enhanced protein turnover with preservation of their lean mass. Collectrin-deficient animals, then, serve as an important in vivo metabolic system in which to study mechanisms of nutrient sensing and regulation of energy homeostasis. Elucidating these mechanistic details could have important therapeutic implications for a wide range of human pathological conditions, such as diabetes and sarcopenia of aging.

Materials and Methods

Generation and maintenance of animals included in this study

The collectrin-deficient mouse line was generated on a mixed genetic background (C57BL/6 and 129/SvEv), as previously described (14). Mice were maintained on either standard rodent control diet containing (wt/wt) 6% fat, 20% protein, and 74% carbohydrate (Harlan Teklad, Indianapolis, IN) or HFD containing (wt/wt) 58% fat, 16% protein, and 26% carbohydrate (Research Diets; New Bronswick, NJ), starting at 2 months of age and continued for 18 wk. All studies were approved by the Duke, Durham Veterans Affairs, and University of Alabama, Birmingham, Medical Centers animal care and use committees.

Metabolic cage studies and assessment of baseline characteristics

Animals were housed singly in metabolic cages (Hatteras Instruments, Cary, NC) with free access to food and water. Body composition was determined using DEXA (Lunar PIXImus; General Electric, Amersham, Buckinghamshire, UK) scans of the subcranial body on anesthetized mice, as previously described (41).

Measurement of serum glucose and insulin levels

Blood was collected via tail vein, and blood glucose was measured with a glucose meter (Ascensia Glucometer Elite; Bayer AG, Leverkusen, Germany). Remaining whole blood was then processed for serum insulin levels (rat insulin ELISA kit with mouse insulin standards; Crystal Chem, Downers, IL).

IPGTT, IPITT, and arginine tolerance test

Mice were fasted for 6 h before testing. Blood samples were collected by tail vein after ip injection of 1.5 or 3 g/kg glucose (Sigma Chemical Co., St. Louis, MO), 0.75 U/kg regular insulin (Eli Lilly, Indianapolis, IN), or 0.3 g/kg l-arginine (Sigma). Changes in blood glucose and serum insulin during IPGTT were determined by calculating AUC between 0 and 60 min using a trapezoidal model.

Insulin secretion from isolated islets

Pancreatic islets were harvested from mice using the Liberase R1 enzyme (Roche Diagnostics, Indianapolis, IN) according to manufacturer protocol, and isolated islets were cultured overnight in RPMI 1640 medium (GIBCO, Carlsbad, CA) containing 8 mmol/liter glucose. After preincubation for 45 min in HEPES balanced salt solution-0.5% BSA, triplicate groups of 20 islets were then incubated at 5.5 or 16.7 mm glucose for 2 h, and cell media samples were used for measurement of insulin secretion, as previously described (42), and normalized to total islet cellular protein.

Histological and morphological analysis of the pancreas and pancreatic islets

Pancreata were removed from mice after maintenance on NC or 18 wk of HFD, embedded in paraffin, and sectioned at three levels separated by 100 μm. Sections were stained with hematoxylin. Immunostaining for insulin was performed per manufacturer protocol with prediluted guinea pig antiinsulin primary antibody (Zymed Laboratories, San Francisco, CA), anti-guinea pig secondary antibody (Vector Laboratories, Burlingame, CA), and Vectastain ABC-alkaline phosphatase detection system (Vector). For β-cell replication, mice were administered 100 mg/kg ip 5-bromo-2′-deoxyuridine (BrdU) solution (Sigma) 6 h before pancreas removal. Pancreatic sections were stained for BrdU using biotinylated monoclonal anti-BrdU antibody and the streptavidin-biotin system (Zymed). Digital images were analyzed using the National Institutes of Health software ImageJ (http://rsb.info.nih.gov/ij/). All morphometric measures were normalized for direct comparisons between groups. For islet number, the total number of islets were counted per section and normalized to the total area of the section as determined using ImageJ. This value was then averaged across sections for each animal. For the relative β-cell mass (β-cell/pancreas area), the total area of all islets in a section was divided by the total pancreas area for that section. This value was again averaged across sections for each animal. For replication data, at least 5000 insulin-positive cells were counted for each treatment, and data are expressed as percentage of the β-cells that stain for BrdU out of the total number of insulin-positive cells per section, as previously described (43).

Measurement of plasma and urine acylcarnitine profiles by mass spectrometry

All analyses employed stable isotope dilution techniques. Plasma FC, acylcarnitine, and creatinine measurements were made by flow injection tandem mass spectrometry using sample preparation methods described previously (44,45). The data were acquired using a Micromass Quattro Micro system (Waters-Micromass, Milford, MA) equipped with a model HTS-PAL 2777 auto sampler (Leap Technologies Carrboro, NC), a model 1525 HPLC solvent delivery system (Agilent Technologies, Santa Clara, CA) and a data system controlled by MassLynx 4.1 operating system (Waters Corp., Milford, MA) at the Stedman Center Mass Spectrometry Lab.

Tissue amino acid quantitation

Tissue was quickly harvested and ground in a liquid nitrogen-chilled mortar and pestle. Approximately 50 mg of the powder was resuspended 20-fold in water, homogenized on ice, sonicated, and spun for 15 min at 4 C, 14,000 rpm (44). Data were normalized to milligrams protein in sample as determined by bicinchoninic protein assay (Sigma). Measurement of amino acids was made from the supernatant after derivatization to their butyl-esters by direct injection electrospray tandem mass spectrometry, using the system described above.

Measurement of serum free fatty acids and ketones, plasma amino acids, and urine 3-methylhistidine

These assays were performed as a core facility service at the Sarah W. Stedman Nutrition and Metabolism Center and the Biochemical and Genetics Laboratory and Mass Spectrometry Facility at Duke University Medical Center.

Statistical analysis

Values for each parameter within a group are expressed as means ± sem. Student’s t test was used for all comparisons between two groups. A P value of 0.05 was used to assess statistical significance. Statistical calculations were done using commercially available software packages (Minitab and NCSS).

Acknowledgments

We thank Dr. Yanqiang Yang for technical assistance with morphometric analysis.

Footnotes

This work was supported by National Institutes of Health (NIH) Grant P01DK58398 to C.B.N., National Kidney Foundation postdoctoral fellowship (to S.M.M.), and Satellite Healthcare Norman S. Coplan Extramural Grant and NIH Grant R01DK065035 (to T.H.L.).

Disclosure Summary: The authors have nothing to disclose.

First Published Online February 26, 2009

Abbreviations: AUC, Area under the curve; BCAA, branched-chain amino acids; BrdU, 5-bromo-2′-deoxyuridine; CoA, coenzyme A; DEXA, dual-energy x-ray absorptiometry; FAO, fatty acid oxidation; FC, free carnitine; HFD, high-fat diet; HNF-1α, hepatocyte nuclear factor-1α; HOMA-IR, homeostasis model assessment of insulin resistance; IPGTT, ip glucose tolerance test; IPITT, ip insulin tolerance test; IR, insulin resistance index; MODY, maturity-onset diabetes of the young; NC, normal chow; RAS, renin-angiotensin system; SC-AC, short-chain acylcarnitines; TCA, tricarboxylic acid.

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